Claude's son François de Lorraine, 2d duc de Guise, 1519-63, became conspicuous, at the accession (1547) of Henry II, as the rival for power of Anne, duc de Montmorency. In the final stages of the Italian Wars, François distinguished himself in the defense of Metz (1552), led the expedition to Italy against King Philip II of Spain, and after the failure of the expedition returned to defend France from English and Spanish attacks; in 1558 he took Calais from the English. With the accession (1559) of the youthful Francis II, who was married to the duke's niece, Mary Stuart, François de Guise and his brother the Charles de Guise, Cardinal de Lorraine, c.1525-1574, were given control of the government.

The brothers' arrogance, their persecution of the Protestants, and their enmity toward the princes of Bourbon and Condé led to the conspiracy of Amboise (see Amboise, conspiracy of), which they suppressed (1560). Shortly afterward, however, the death of Francis II deprived the Guises of power; Catherine de' Medici, as regent, dominated the government. As a result, in 1561 the duke joined with Montmorency and Marshal Saint-André in the so-called triumvirate, which, at the head of the Catholic party, opposed both the Huguenots and the tolerant policy of the regent. The murder of Protestants at Vassy by Guise's troops brought about the outbreak of the Wars of Religion (1562-98; see Religion, Wars of), and Guise took the field against the Huguenots. Victorious at Dreux (1562), he was assassinated while preparing to attack Orléans.

The Cardinal de Lorraine was largely responsible for the persecution of the Protestants during the reign of Francis II. At the Colloquy of Poissy (1561) he defended Catholicism against Theodore Beza; at the Council of Trent (1562-63) he at first upheld the independence of the Gallican church but later reversed his position and attempted to have the decrees of the council proclaimed in France. He subsequently negotiated with Philip II of Spain for Spanish support of the Catholic cause in France. After the downfall of Michel de L'Hôpital, Charles temporarily returned to power until 1570. He was the most consummate politician in his family and a master of intrigue.

Charles's nephew Henri de Lorraine, 3d duc de Guise, 1550-88, son of François, fought in the Wars of Religion and cooperated with Catherine de' Medici in planning the massacre of the Huguenots on Aug. 24, 1572 (see Saint Bartholomew's Day, massacre of). After the peace of 1576 he formed the Catholic League (see League), and King Henry III, although secretly afraid of the League, became its nominal head. After the death of Francis, duke of Alençon and Anjou (1584), Henri de Lorraine revived (1585) the League in opposition to the Protestant Henry of Navarre (later King Henry IV), who had become heir presumptive to the throne.

War broke out between the League and Henry of Navarre. Although the king was the nominal head of the League, he was overshadowed by the immensely popular de Guise, who had designs on the throne. In May, 1588, when de Guise returned to Paris, the Parisians revolted against the king on the Day of the Barricades (May 12). However, instead of taking the throne Guise permitted Henry III to escape, and the king named him lieutenant general of France. Later in the same year, however, the king brought about his assassination.

Henri's brother Louis de Lorraine, Cardinal de Guise, 1555-88, was killed at the same time as Henri. After their deaths the leadership of the League devolved upon their brother, Charles, duc de Mayenne. Henri was succeeded by his son Charles de Lorraine, 4th duc de Guise, 1571-1640.

Henri de Lorraine, 5th duc de Guise, 1614-64, son of the 4th duke, was archbishop of Reims but became duke after the death of his older brother (1639) and of his father. He conspired (1641) against Cardinal Richelieu and was forced to live in exile for a time in Flanders. In 1647 he took part, as representative of the house of Anjou, in the insurrection at Naples against Spanish rule. Captured by the Spanish (1648), he was a prisoner until 1652. He made a new attack on Naples in 1654, then returned to Paris, where, as grand chamberlain, he played a prominent role in the social life of the court. He was succeeded by his nephew, Louis Joseph de Lorraine, 6th duc de Guise, 1650-71. With François Joseph de Lorraine, 7th duc de Guise, 1670-75, son of the 6th duke, the line came to an end.