We examined the effects of 5-(N,N-hexamethylene)amiloride (one of the Na+-H+ exchange blockers, HMA) and amiloride (AM) on action potentials (APs), intracellular Na+ activity, and pH using conventional and double-barreled ion-selective microelectrodes in guinea pig papillary muscle in vitro. Papillary muscle preparations were superfused with HEPES-buffered solution, and intracellular Na+ (a° +) and H+ (intracellular pH, pHi) activities were measured in quiescent preparations without stimulation. HMA at a concentration of 1 μM began to induce prolongation of action potential duration (APD) and at concentrations > 10 μM induced a decrease in action potential amplitude (APA), depolarization of resting membrane potential (RMP), prolongation of APD and depression of the maximum upstroke velocity (Vmax). HMA exerted dose-, time-, and rate-dependent reduction in Vmax. AM began to prolong APD at a concentration of 10 μM and at 1 mM induced depolarization of RMP, decreased Vmax and induced significant prolongation of APD. HMA (100 μM) induced a decrease in a° + by 2–3 mM, but exerted no effects on pH; in normal Tyrode's solution. Under conditions of intracellular acidosis induced by exposure to K +-free solution, HMA produced a further decrease in pHi. Our results provide direct evidence that HMA has a depressant action on cardiac Na + channels and prolongs AP at concentrations that presumably affect Na+-H+ exchange in cardiac muscle.