Phorbol-12, 13-dibutyrate (PDB) reduced concentration-dependently the contractile force of guinea-pig papillary muscles (EC50 1.07 mumol/l) while phorbol-12-myristate-13-acetate (PMA) was ineffective. The protein kinase C inhibitors staurosporine (0.1 mumol/l) and polymyxin B (70 mumol/l) did not antagonize the negative inotropic effect of PDB. Neither PMA nor PDB, in concentrations up to 30 mumol/l, caused significant changes of the maximum depolarization velocity, the action potential duration or the functional refractory period in intact papillary muscles. In isolated ventricular cardiomyocytes the inward calcium current was halved by either 1 mumol/l PDB or 10 mumol/1 PMA. PKC inhibitors attenuated, but could not completely abolish this effect of the phorboles. It is concluded that the negative inotropic effect of PDB is caused by a reduction of the slow inward calcium current and that this inhibition is, for the greater part, not mediated by an activation of protein kinase C.