S100C (S100A11, calgizzarin) inhibits the actin-activated myosin Mg2+-ATPase activity of smooth muscle in a dose-dependent manner: its half-maximal effect occurs at a S100C/actin molar ratio of 0.05 and its maximal effect occurs at a ratio of 0.20. Furthermore, S100C was found to bind to actin with a stoichiometry of 1:6-7 in the presence of Ca2+, with an affinity of 1 x 10(-6) M determined by cosedimentation assays. Other Ca2+-binding proteins such as S100A1, S100A2, S100B, and calmodulin did not inhibit actin-activated myosin Mg2+-ATPase activity. Calmodulin, S100A1, and S100B reversed the inhibitory effect of calponin in a Ca2+ dependent manner, S100A2 had no effect, and S100C had additional inhibitory effects. The results suggest that S100C might be involved in the regulation of actin-activated myosin Mg2+-ATPase activity through its Ca2+-dependent interaction with actin filaments.