The cellular and subcellular distribution of the S-100 protein in rabbit and rat central nervous system was studied both quantitatively and qualitatively. Microcomplement fixation estimations on bulk-prepared neuronal and glial cells showed at least five to six times higher amounts of water-soluble S-100 in glial cell-enriched fractions as compared to fractions enriched in neuronal perikarya. Nerve cell fractions contained a higher percent of tissue-bound S-100 protein. High levels of S-100 protein were found in mitochondria and soluble protein fractions. Immunoelectron microscope investigations demonstrated S-100 protein in neuronal structures, such as the postsynaptic membrane and part of the plasma membrane. Among glial cells the astrocytic filaments contained high levels of S-100 protein. S-100 was also found in most subcellular membranes of astrocytes and oligodendrocytes.