Cytoplasmic and membrane-bound S-100 proteins were purified to homogeneity from bovine and rat brain. Cytoplasmic and membrane-bound S-100 from single species are identical by immunological, electrophoretic, spectrophotometric, and functional criteria. Cytoplasmic and membrane-bound S-100 from bovine brain consists of nearly equal amounts of S-100a and S-100b, whereas cytoplasmic and membrane-bound S-100 from rat brain consists mostly of S-100b. The functional role of membrane-bound S-100 remains to be elucidated.