The cardioprotective activity of propranolol is believed to be independent of its beta-adrenoceptor antagonistic effect. Propranolol exerts this effect through a direct effect on the cardiac muscle, but the precise mechanism remains unclear. In this work, we demonstrated that propranolol binds to S100ao and S100L proteins with ED50 of approximately 1.0 microM without cation dependency and that this binding changes the conformation of these S100 proteins. Propranolol, however, was found to bind to and to change the conformation of S100C protein in the presence of Mg2+ or Zn2+ with ED50 of approximately 1.0 microM. No change was observed in the presence of Ca2+. Moreover, in the presence of Mg2+, the ED50 of L- and D- propranolol were approximately 0.8 and 2.0 microM, respectively. This study demonstrated for the first time, that the S100 proteins of the cardiac muscle are intracellular targets of propranolol, and that Mg2+ is a modulator of the cardioprotective activity of S100C protein.