S100 proteins are EF-hand calcium-binding protein highly preserved during evolution present in both neuronal and non-neuronal tissues of the higher vertebrates. Data about the expression of S100 protein in fishes are scarce, and no data are available on zebrafish, a common model used in biology to study development but also human diseases. In this study, we have investigated the expression of S100 protein in the central nervous system of adult zebrafish using PCR, Western blot, and immunohistochemistry. The central nervous system of the adult zebrafish express S100 protein mRNA, and contain a protein of approximately 10 kDa identified as S100 protein. S100 protein immunoreactivity was detected widespread distributed in the central nervous system, labeling the cytoplasm of both neuronal and non-neuronal cells. In fact, S100 protein immunoreactivity was primarily found in glial and ependymal cells, whereas the only neurons displaying S100 immunoreactivity were the Purkinje's neurons of the cerebellar cortex and those forming the deep cerebellar nuclei. Outside the central nervous system, S100 protein immunoreactivity was observed in a subpopulation of sensory and sympathetic neurons, and it was absent from the enteric nervous system. The functional role of S100 protein in both neurons and non-neuronal cells of the zebrafish central nervous system remains to be elucidated, but present results might serve as baseline for future experimental studies using this teleost as a model.