Cholinergic innervation of the rat cerebellum was investigated immunohistochemically by using a monoclonal antibody against choline acetyltransferase. Immunoreactive structures included: 1) a subpopulation of mossy fibers and glomerular rosettes; 2) thin varicose fibers, which were closely associated with the Purkinje cell layer and also found in the molecular layer; and 3) relatively dense networks of varicose fibers distributing in the cerebellar nuclei. Quantitative analysis indicated that a great many immunoreactive rosettes were localized in lobules IXc and X, although their density in lobule X was approximately four times that in the lobule IXc. A considerable number of immunoreactive structures were also present in all other lobules. In the hemispheres they were confined to a zone immediately beneath the Purkinje cell layer, whereas in the vermis they were scattered throughout the granular layer. Most of the immunoreactive fibers found in the molecular layer coursed toward the pial surface and were distributed within the inner half of the molecular layer. In the cerebellar nuclei, portions of the medial nucleus and magnocellular portion of the lateral nucleus had moderately dense networks of immunoreactive fibers, whereas loose networks of fibers were observed in the posterior interposed nucleus. Other parts of the cerebellar nuclei contained a smaller number of varicose fibers.