With the use of microelectrodes, intracellular pH (pHi), surface pH (pHs), and intracellular Na+ activity (aiNa) were measured in isolated guinea pig papillary muscles during normal superfusion and during a reversible condition of simulated ischemia. Acid loading by NH+4 prepulse or by CO2-HCO3- addition during superfusion with pH 7.4 solutions caused internal acidification followed by a recovery of pHi, which could be inhibited by amiloride. pHi recovery was associated with an amiloride-sensitive peak rise of aiNa and membrane hyperpolarization, indicative of Na(+)-H+ exchange. Peak increase of aiNa was absent if the pH of the superfusion solution was concomitantly lowered. Imposed ischemia after control superfusion caused membrane depolarization and acidification of pHi and pHs. The change of pHs consistently was larger than that of pHi. aiNa decreased from 5.5 to 4.6 mM after 10-min ischemia. Enlarging the pHi (and pHs) decrease in ischemia by prior reduction of the tissue buffer capacity (CO2-HCO3(-)-free superfusion) was unable to induce a rise of aiNa during the subsequent ischemic period. Amiloride had no significant effect on aiNa during ischemia. It is concluded that the important acidification of pHs reduces the rate of pHi regulatory Na(+)-H+ exchange and thereby contributes to a longer maintenance of the Na+ electrochemical gradient in ischemic cardiac muscle.