Serotonin (5-HT) uptake and release were analyzed in cerebellar fractions. Cerebellar, P2 fractions and molecular layer homogenates are able to accumulate exogenous 5-HT through an Na-dependent, high-affinity transport system. Molecular layer 5-HT uptake, however, was totally dependent upon Na+, whereas the accumulation of the amine by P2 fractions from whole cerebellum was also carried out by an Na-independent transport system. Ca++-dependent release of recently accumulated 5-HT was observed in both P2 fractions and molecular layer homogenates when depolarizing conditions were used. This release was blocked by ruthenium red and Mg++, two inhibitors of the Ca++ entry to the nerve endings induced by depolarization. The molecular layer homogenates released twice as much 5-HT as did cerebellar P2 fractions, indicating that serotoninergic boutons in the cerebellar cortex might be located in the molecular layer. Our results suggest that 5-HT might play a transmitter role in the cerebellum.