We report that bepridil, a Ca++ channel blocker and calmodulin antagonist, which has been shown to enter myocytes, stimulates the mechanical and biochemical activity of cardiac myofilaments. Bepridil increased significantly the level of Ca++-dependent actomyosin Mg++-ATPase activity of myofibrils and the submaximal force developed by chemically skinned trabeculae of pig heart. In the range of concentrations (10-100 microM) in which bepridil showed this stimulatory activity, diltiazem and verapamil were without effect. The effect of bepridil on myofilament force and ATPase activity was higher at relatively low free Ca++ concentrations, and myofibrils lacking troponin-tropomyosin were not affected by bepridil. Associated with the stimulation of force and ATPase activity by bepridil was an increase in the amounts of Ca++ bound to troponin C (TnC). That bepridil stimulates TnC Ca++ binding was also shown in experiments using pure TnC labeled with 2-(4'-iodoacetamidoanilo)naphthalene-6-sulfonic acid, a fluorescent probe that reports Ca++ bound to the single "regulatory" site. Effects of bepridil on the fluorescence of a felodipine-cardiac TnC complex indicate that bepridil binds to TnC over the same range of doses where it affects myofilament activity. Our results indicate that the inotropic action of bepridil may result from a net response of heart cells to influences on the delivery of Ca++ to the myofilaments and their response to Ca++.