The distribution of serotonin immunoreactivity in the rat cerebellum was studied using the indirect antibody peroxidase-antiperoxidase (PAP) technique of Sternberger. Furthermore, the origin of these chemically defined cerebellar afferents was studied using a procedure which combines the retrograde transport of horseradish peroxidase (HRP) with the PAP technique. Serotoninergic fibers and varicosities distribute throughout the cerebellar cortex. However, within the cortex there are density variations in the distribution of this indoleamine to the granule cell and molecular layers as well as differences in the spatial orientation of labeled elements, especially in the latter lamina. Serotonin-positive fibers are also present in the Purkinje cell layer. Some of the fibers pass from this layer into the overlying molecular layer while others form a plexus around the somata of Purkinje cells. Subsequent to injections of HRP into the vermis and immediately adjacent portions of the cerebellar cortex, several reticular and raphe nuclei (n.) were found to project to the cerebellum including the paramedian reticular nucleus, n. raphe pallidus, n. raphe obscurus, n. raphe magnus, n. reticularis gigantocellularis, n. reticularis paragigantocellularis, n. pontis oralis, n. reticularis tegmenti pontis and n. centralis superioris. Double-labeling experiments, however, reveal that the neurons giving rise to serotoninergic afferents to the cerebellum are located almost exclusively in the n. reticularis gigantocellularis, the n. reticularis paragigantocellularis and the n. pontis oralis. In conclusion, the findings of the present study further support the view that the cerebellar cortex is not uniform in its histological structure. Although serotoninergic elements are distributed throughout the cerebellar cortex, there are lobular variations in the laminar distribution of this indoleamine. These data suggest that serotonin may be exerting its physiological effect on different populations of cortical neurons in different lobules. Thus this putative neurotransmitter may play different roles in the circuitry of the cerebellum in disparate regions of the cortex. Further, the data obtained in the double-label experiments demonstrate a fairly restricted origin for serotoninergic afferents in the medullary and pontine reticular formation. Moreover, the majority are not located in the raphe nuclei.