The distribution of protein kinase C (PKC) subspecies and their colocalization with neurotransmitters were examined in the rat striatum and substantia nigra (SN), using immunocytochemistry. The alpha- and beta I-PKC immunoreactivies were seen predominantly in the perikarya of the neurons in the striatum and SN. In contrast, the beta II- and gamma-PKC immunoreactivities were abundant in both the perikarya and the neuropils in the striatum and only in the neuropils in the SN. From electron microscopic studies, the alpha- and beta I-PKC immunoreactivities were seen adjacent to the plasma membrane, while the beta II-PKC immunoreactivity was observed in the cytoplasm around the Golgi complex. The gamma-PKC immunoreaction was dense throughout the cytoplasm. The double-staining and lesion studies revealed that the alpha-PKC-immunopositive neurons in the striatum were intrinsic cholinergic neurons, and that most of the alpha-PKC-immunoreactive neurons in the SN were dopaminergic neurons. The beta I-PKC-immunoreactive neurons were intrinsic GABAergic neurons in the striatum. Moreover, most of the beta II- and gamma-PKC-immunoreactive neurons were medium-sized neurons projecting to the SN, and over 90% of GABAergic neurons in the caudate-putamen contained beta II-PKC. The beta II-PKC-immunoreactive neurons showed no gamma-PKC immunoreactivity, and the gamma-PKC-immunoreactive neurons were not beta II-PKC immunoreactive. These findings suggest that alpha-PKC is related to the function of the nigral dopaminergic and the striatal cholinergic neurons, and that the beta I-PKC is involved in the function of the striatal intrinsic GABAergic neurons. The beta II- and gamma-PKC may also modulate a specific neuronal function in the striatonigral system.