Choline acetyltransferase (CAT) activity was measured in the cat brain using a microdissection of the structures from frozen slices and an extremely sensitive radioisotopic assay for the enzyme. About 20 cerebral regions were chosen for study because of their role in sensorimotor integration. Highest CAT activity was found in the basal ganglia, followed by the thalamus and the cerebral cortex. The cerebellum had the lowest level of CAT. Special detailed studies were made in the substantia nigra, nucleus ventralis lateralis of the thalamus, areas 4 and 6 of the cerebral cortex, the red nucleus in the mesencephalon and the deep cerebellar nuclei in order to define the intranuclear distribution of their cholinergic innervation.