Available data are conflicting as regards the occurrence of Ca2+ and Zn2+ binding S100 proteins in neurons of mammalian brain. Here the localization and expression of S100 was re-investigated using several different antibodies and in situ hybridization. A map is provided for the distribution of two classes of S100-positive neuron populations in the adult rat CNS. "Persistently S100-positive" neurons had large size, were strongly immunoreactive and were mainly distributed in the nuclei of the lower brainstem and cerebellum. "Variably S100-positive" neurons were preferentially found in the forebrain of rats older than 90 days and were especially numerous in limbic regions. The S100-immunoreactivity in these neurons was moderately intense, occurred with high interindividual variation and appeared related to function as suggested by variations due to anesthesia. The expression of S100 mRNA in neurons was re-investigated at high spatial resolution with non-radioactive in situ hybridization using an oligonucleotide specific for S100 beta-mRNA. Expression of S100 was demonstrated in astrocytes and in those neuron populations which were also strongly S100-immunoreactive. No expression of S100 beta message was seen in weakly immunoreactive neurons, b but this may be due to low sensitivity of the techniques used. The data suggest that the S100 proteins are synthesized in all astrocytes and in distinct subpopulations of neurons in rat brain. These neurons show a characteristic topography and vary in S100 expression probably due to their function and maturation.