E-4031 and related methanesulfonanilide class III antiarrhythmic drugs block I(Kr), a cardiac delayed rectifier K+ current. The current-voltage relationship of I(Kr) exhibits rectification; currents progressively decline in magnitude at test potentials >0 mV. Whole-cell voltage-clamp techniques were used to determine whether rectification results from block of channels by intracellular Mg++. The properties of E-4031-sensitive current were compared in guinea pig ventricular myocytes internally perfused with either a nominally Mg++ -free solution or with a solution containing 1mM Mg++. Based on an envelope of tails test, we conclude that inward rectification of guinea pig I(Kr) is due to a voltage-dependent gating mechanism and does not result from block of the channel by intracellular Mg++. Under normal physiologic conditions, E-4031 is a specific blocker of I(Kr). However, in the absence of intracellular Mg++, E-4031 also partially blocks I(Ks). Block of I(Ks) is prevented by prior treatment of cells with isoproterenol, which suggests that E-4031 only blocks unphosphorylated I(Ks) channels in the absence of intracellular Mg++.