Superfusion of guinea pig papillary muscles with Tyrode's solution that contained 0.3% to 10% dimethyl sulfoxide (DMSO) caused a small hyperpolarization, a prolongation of the action potential (e.g., 4%, 15% and 33% prolongation with 1%, 5% and 10% DMSO, respectively) and a reduction (< or = 14%) in the maximal upstroke velocity (Vmax). Action potential prolongation was also induced by DMSO in muscles treated with the inorganic Ca++ channel blocker Co++ (5 mM) and in those depolarized with 24 mM K(+)-containing Tyrode's solution. In the latter case, there was no significant change in Vmax. In voltage-clamp experiments, superfusion of guinea pig ventricular myocytes with 0.1% to 10% DMSO solution had little effect on Ca++ and inward-rectifier K+ currents, moderately inhibited Cl- and Na+ currents, partially inhibited Na+ pump current and markedly depressed delayed-rectifier K+ current. The cell water shrinkages measured in muscles and myocytes superfused with DMSO solution appear to be only partially responsible for the solvent's effects. Other mechanisms that may be of greater importance in explaining the action of DMSO include direct block of channels by DMSO molecules and hindrance of channel hydration and opening related to osmotic stress.