We have investigated the effect of antiarrhythmic drugs on the increased potassium conductance induced in isolated adult rat heart cells by ATP depletion. The rate of86Rb uptake in the presence of ouabain was used as a measure of potassium conductance. Treatment of cells with rotenone plusp-trinuoromethoxyphenylhydrazone (FCCP) rapidly depleted ATP levels and strongly stimulated the rate of86Rb uptake. The stimulated uptake and the ATP depletion were inhibited by oligomycin; thus, the uptake was not induced by roteuone plus FCCP directly. The stimulated uptake, but not the ATP depletion, was inhibited potently by glyburide (ICJO, 38.3 nM), quinidine (IC50, 2.7 μM), verapamil (IC50, 4.5 μM), and amiodarone (IC50, 19.1 μM). The stimulated uptake was also inhibited by tetraethylammonium ion and by 4-aminopyridine but not by tetrodotoxiii or manganese. We conclude that 1) the stimulated86Rb uptake is measuring ATP-sensitive potassium channel activity, 2) the ATP-sensitive potassium channel is strongly inhibited by quinidine, verapamil, and amiodarone, and 3) this inhibition may contribute to the antiarrhythmic action of these drugs.