1. The effect on intracellular pH (pHi) and intracellular Na+ activity (aNai) of exposure to hyperosmolar solutions was investigated in guinea-pig ventricular muscle using ion-sensitive microelectrodes. 2. Exposure of tissue to solution made hyperosmolar by the addition of 100 mM-sucrose produced an intracellular alkalinization of 0.10 pH units and hyperpolarization of the membrane potential. 3. When extracellular Na+ was reduced to 15 mM by substitution of NaCl with choline chloride, exposure to hyperosmolar solutions caused a decrease in pHi. Identical experiments using LiCl as the sodium substitute resulted in an increase in pHi of a magnitude similar to that seen at physiological Na+ levels. 4. In the presence of 50 microM-5-(N,N-dimethyl)amiloride (DMA), an inhibitor of Na(+)-H+ exchange, pHi decreased upon exposure to hyperosmolar solution. 5. The recovery of pHi from an intracellular acidosis (induced by brief exposure to NH4Cl) was enhanced in hyperosmolar solution when compared to recovery in isosmolar solution. This enhancement was observed even when aNai was markedly elevated (greater than 25 mM) by inhibition of the Na(+)-K+ pump. 6. There was an increase in aNai during exposure to hyperosmolar solutions. When the Na(+)-K+ pump was inhibited with dihydro-ouabain a component of this increase in aNai was sensitive to DMA. 7. We conclude that exposure of cardiac tissue to hyperosmolar solutions results in an intracellular alkalosis due to activation of the sarcolemmal Na(+)-H+ exchanger. Such changes should be considered when exposure to hyperosmolar solutions is used in the study of excitation-contraction coupling and cardiac muscle mechanics.