Phenamil, a phenyl-substituted amiloride derivative and a potent inhibitor of epithelial-type sodium channels, produces significant prolongation of the action potential in isolated ventricular preparations, a tissue in which epithelial-type sodium channels have not been reported. Experiments were therefore carried out to examine the possible electrophysiological effects of phenamil on whole-cell ionic currents recorded in voltage-clamped guinea pig ventricular myocytes. At higher concentrations than those required to inhibit the epithelial sodium channels, phenamil (10-60 microM as compared to nanomoles for epithelial sodium channels) significantly increased the action potential duration in ventricular myocytes. It also induced a slow progressive depolarization which was followed eventually by oscillatory potentials near -35 mV. Higher concentrations of phenamil accelerated these changes. These effects were partially reversible. Voltage-clamp experiments using slow voltage ramps from -140 mV to +60 mV or step protocols from -120 mV to +30 mV revealed a prominent (approximately 70% at -120 mV) inhibitory effect of phenamil (50 microM) on the inwardly rectifying potassium current. Phenamil (50 microM) had little effect on the delayed rectifier potassium currents recorded by using long (5 sec) voltage steps from -40 mV to +60 mV. In the absence of K+, calcium current (L-type) was not affected by phenamil at concentrations up to 100 microM. Our data support the concept that phenamil-induced action potential prolongation in guinea pig cardiac myocytes may be produced by inhibition of inwardly rectifying potassium current.