Microtubule-associated proteins (MAPs) of brain microtubules exhibit an ATPase activity which is markedly enhanced by tubulin and Ca(2+). Addition of S100 protein decreased the tubulindependent Ca(2+)-ATPase activity by about 85%, but did not affect the activity without tubulin. The inhibition by S100 protein was concentration-dependent and the apparent K(m) value for ATP was not altered. A large amount of tubulin restored the inhibition, indicating that S100 protein acts through the binding to the tubulin molecule. Zn(2+), which can bind both microtubule proteins and S100 protein, had little effect on the inhibitory action of S100 protein. The ATPase inhibition by S100 protein was partially restored by chlorpromazine or vinblastine. S100a is more effective than S100b on the inhibitory effect of tubulin-dependent ATPase activity. The results suggest that S100 protein may function as a regulatory factor of ATPase in brain microtubules.