The mts1 gene codes for a 9 kDa protein belonging to the S100 subfamily of Ca2+-binding proteins and is known to play a role in metastasis. Its role in metastasis may be through cellular locomotion, as transfection of mts1 into mouse mammary adenocarcinoma cells increases cellular motility in modified Boyden chemotaxis chambers. The Mts1 protein interacts with nonmuscle myosin II in the presence of Ca2+ with an affinity of approximately 7.9 x 10(4) M-1 and an approximate stoichiometry of 3 mol of Mts1/mol of myosin heavy chain. No interaction was found with myosin I or myosin V. The binding site of Mts1 on myosin is in the rod region, particularly to the light meromyosin portion of the rod. To understand the mechanism by which Mts1 alters cellular motility, we examined its effect on myosin structure and activity. Cosedimentation analysis and electron microscopy suggest that Mts1 destabilizes myosin filaments. In the presence of Ca2+, Mts1 inhibits the actin-activated MgATPase activity of myosin in vitro. The data demonstrate an effect of Mts1 on both myosin structure and function, and suggest a route through which Mts1 affects motility as well as metastasis.