Thrombin can activate the plasma membrane Na(+)-H+ exchanger in a variety of noncardiac cells. We have studied (1) the effect of thrombin on the activity of the sarcolemmal Na(+)-H+ exchanger in freshly isolated quiescent ventricular myocytes from the adult rat heart and (2) the signaling mechanism(s) underlying any effect. Reverse-transcription polymerase chain reaction analysis revealed thrombin receptor mRNA expression in a myocyte-enriched cell preparation. As an index of Na(+)-H+ exchanger activity, acid efflux rates (JHS) were determined in single myocytes (n = 4 to 11 per group) loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after two consecutive intracellular acid pulses (induced by transient exposure to 20 mmol/L NH4Cl) in bicarbonate-free medium. At a pHi of 6.9, JH did not change significantly during the second pulse relative to the first in control cells. However, when the second pulse occurred in the presence of 0.2, 1, or 5 U/mL thrombin, JH increased by 30%, 62% (P < .05), and 87% (P < .05), respectively. A hexameric thrombin receptor-activating peptide (SFLLRN) mimicked the effect of thrombin and increased JH by 73% (P < .05) at 25 mumol/L. In contrast, an inactive control peptide (FLLRN) was without effect at 25 mumol/L. In cells pretreated with 100 nmol/L GF109203X or 5 mumol/L chelerythrine (protein kinase C inhibitors), neither 5 U/mL thrombin nor 25 mumol/L SFLLRN produced a significant increase in JH. In the presence of 10 mumol/L HOE-694 (a Na(+)-H+ exchanger inhibitor), pHi did not recover after an acid load, even during exposure to 5 U/mL thrombin or 25 mumol/L SFLLRN, confirming that the Na(+)-H+ exchanger was the primary acid efflux mechanism under the conditions used. Neither 5 U/mL thrombin nor 25 mumol/L SFLLRN affected resting pHi and Ca2+ or background acid loading. We conclude that (1) adult rat ventricular myocytes express a functional thrombin receptor, whose stimulation results in increased activity of the sarcolemmal Na(+)-H+ exchanger, and (2) this effect appears to occur through a protein kinase C-mediated mechanism.