The modulation of the transient outward K+ current (Ito) by divalent cations was studied in enzymatically isolated rat ventricular myocytes with the whole cell patch-clamp technique. At holding potentials negative to -70 mV, 1 mM Cd2+ suppressed Ito, whereas, at potentials positive to -50 mV, the current was augmented. These effects were caused by shifts in the voltage dependence of both activation and inactivation of Ito toward more positive potentials. Cd2+ also slowed the activation kinetics of Ito by shifting the voltage dependence of its rate of activation, but the rate of inactivation was unaffected. Other divalent cations produced similar shifts but at markedly different concentrations. Thus, in the millimolar range, a rightward shift of approximately 20 mV was produced by 3 Co2+, 5 Ni2+, and 10 Ca2+, whereas 10 microM concentrations of Cu2+ and Zn2+ produced equivalent shifts. Similar effects were seen in hippocampal neurons with micromolar concentrations of Zn2+. Thus divalent cations have marked and specific effects on the kinetics and voltage dependence of Ito and may serve as a regulatory mechanism in its activation, particularly in cells with resting potentials positive to -60 mV.