Cholinergic innervation of the human cerebellum was investigated immunocytochemically by using a polyclonal rabbit antiserum against choline acetyltransferase. Immunoreactive structures were found throughout the cerebellar cortex but were localized predominantly in the vermis, flocculus, and tonsilla. These included 1) a population of Golgi cells in the granular layer; 2) a subpopulation of mossy fibers and glomerular rosettes; 3) thin, varicose fibers closely associated with the Purkinje cell layer and the molecular layer; and 4) a relatively dense network of fibers and terminals contributing to the glomerular formations in the granular layer. In the cerebellar nuclei, some cells stained positively for choline acetyltransferase, and a terminal field pattern could be detected with a distinct but sparse network of varicose fibers. Acetylcholine appears to be a primary transmitter in the vestibulocerebellar pathways at several levels, which may account for the potent effects of muscarinic antagonists in diminishing vestibular vertigo in humans.