In the presence of Ca2+, S-100 protein suppressed microtubule assembly in vitro in a concentration-dependent fashion and the inhibition was restored by adding EGTA. The binding of microtubule proteins to S-100 protein was examined using affinity chromatography on S-100 protein attached to Sepharose 4B. When tubulin or microtubule-associated proteins (MAPs) were applied to an S-100 protein-Sepharose 4B column, they were bound to the column in the presence of Ca2+, but were not relased by adding EGTA. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated that the proteins bound to the column, which were eluted with 0.5 M KCl plus EGTA, consisted of tubulin or tau proteins.