The cholinergic innervation of the cerebellar cortex of the rat was studied by immunohistochemical localization of choline acetyltransferase, radiochemical measurement of ChAT activity, and double labeling of ChAT-positive neurons with HRP injected into the cerebellum. ChAT immunohistochemistry revealed large mossy fiber rosettes as well as finely beaded terminals with different morphological characterization, laminar distribution within the cerebellar cortex, and regional differences within the cerebellum. Large "grapelike" ChAT-positive mossy fiber rosettes that were distributed primarily in the granule cell layer were concentrated, but not exclusively located, in three separate regions of the cerebellum: (1) the uvula-nodulus (lobules 9 and 10); (2) the flocculus, and (3) the anterior lobe vermis (lobules 1 and 2). Regional differences in ChAT-positive afferent terminations in the cerebellar cortex demonstrated by immunohistochemistry were confirmed by regional biochemical measurements of ChAT activity. Using ChAT immunohistochemistry in combination with HRP injections into the uvula-nodulus, we have studied the origin of the cholinergic projection. The caudal medial vestibular nucleus and to a lesser extent the nucleus prepositus hypglossus contain ChAT-positive neurons that were double labeled following HRP injections into the uvula-nodulus. We conclude that (1) there is a prominent cholinergic mossy fiber pathway to the vestibulocerebellum, (2) this pathway originates primarily in the caudal third of the medial vestibular nucleus, and (3) this cholinergic pathway likely mediates secondary vestibular information related to postural adjustment.