Distinct expression of protein kinase C (PKC) subspecies in the central nervous system suggested that each subspecies has a distinct neural function in the processing and modulation of a variety of physiological responses to external signals. In this study, the cellular and subcellular distributions of beta I-, beta II- and gamma-subspecies of PKC were demonstrated by using subspecies-specific antibodies in the rat spinal cord. By light microscopy both gamma- and beta II-subspecies immunoreactivities were found only in neurons of the substantia gelatinosa and axons of the dorsal corticospinal tract in the spinal cord. Use of a double staining method, however, revealed that beta II-subspecies immunoreactivity was localized in the outer part of the lamina II, whereas gamma-subspecies immunoreactivity was found in the inner part of lamina II. Immunoreactive neurons containing beta I-subspecies were scattered in the substantia gelatinosa. Beta I-subspecies immunoreactivity varied in neuronal types. Furthermore, electron microscopic analysis clearly showed the subcellular distribution of these subspecies to be different from one another. Dense gamma-subspecies immunoreactivity was found in the cytoplasm except within cell organelles of the perikarya and dendrites. Some nuclei were stained as strongly as the cytoplasm and others were stained less heavily. The nucleoli had faint or no immunoreactivity. Reaction products of beta II-subspecies were located against the inner plasma membrane but not seen in the nuclei or nucleoli. Beta I-subspecies immunoreactivity appeared to be associated with the Golgi complex. No immunoreactive products of any PKC subspecies were detected in the presynaptic terminals. The different patterns of expression described above imply that individual PKC subspecies may have a specific function in modulating the neuronal activity in the different neurons of the spinal cord.