Two calcium-binding sites of the Mts1 protein, a member of S-100 protein family, were distinguished with the Fluo-3 fluorescent technique. The geometric mean of the apparent dissociation constant (Kd) for these two sites is 2.6 microM; the Hill coefficient (nH) is 0.98. In the presence of a novel target protein p37, isolated from the mouse adenocarcinoma cell line CSML-100, Mts1 binds Ca2+ ions with higher affinity and with strong positive cooperativity (Kd = 0.2 microM, nH = 1.91). Interaction of Mts1 with p37 is confirmed by the fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS). Reaction with TNS shows that p37 interacts with the hydrophobic site of Mts1 which is exposed due to the binding of Ca2+ ions.