Lashley's major work was in the area of brain function and learning, using learning and discrimination tasks to study the effects of ablation at sites in the rat's brain. His academic training was originally in zoology, but he became interested in psychology while researching for his doctorate. He worked with J.B. Watson and was strongly influenced by the behavouristic approach. During his postdoctoral period he started to work with S.I. Franz at St Elizabeth's Hospital, Washington DC. He studied some neurological cases there, including patients who had received spinal injuries resulting in limb differentiation. His paper on these is still well cited by researchers in motor behaviour, and he is credited with formulating the concept of the motor programme. At this time he also acquired the necessary techniques for performing ablation studies with rats, which allowed him to look for the neural basis of the connectionism of behaviouristic theory. He attempted to find localized sites in the brain specific to various visual discriminations involved in maze learning and these results suggested field theory. It has been said that the discriminations required in his tasks involved cues in modalities other than vision, which would lead to cautious acceptance of this conclusion.

He proposed two influential principles. The first, mass action, is the mediation of certain learning tasks by the cortex as a whole. Greater localization than he suggested has subsequently been found, but mass action events have been found within localized areas. The second is equipotentiality or the ability of certain parts of a sensory system to assume the functions of the other parts. This has subsequently been supported.

Lashley also made a major contribution to neuroanatomy by tracing the connections from the retina to the lateral geniculate nucleus and then to the cortex, and by demonstrating the point-to-point projections later confirmed with more sophisticated techniques.