The role of the Na(+)/H(+) exchanger in ischemia, reperfusion, and preconditioning was investigated in isolated perfused rat hearts. Contractile function, [Na(+)](i), and pH(i) were measured; ischemic damage was assessed by the recovery of developed pressure (DP) on reperfusion. After 30 minutes of ischemia, DP recovered to only 14+/-4% of preischemic control. In contrast, after preconditioning (3x5-minute periods of ischemia) followed by 30 minutes of ischemia, DP recovered to 75+/-4%. Hearts treated with the Na(+)/H(+) exchange inhibitor 5-(N-methyl-N-isobutyl)amiloride (MIA) also showed an enhanced recovery after ischemia (DP 62+/-9%). Treatment with a low concentration of tetrodotoxin (TTX, 100 nmol/L), which blocks the persistent component of the Na(+) current, had a small beneficial effect on recovery (DP 37+/-8%). Thirty minutes of ischemia caused a small [Na(+)](i) rise (3.2+/-0.9 mmol/L); reperfusion resulted in a further [Na(+)](i) increase (+11.9+/-2.5 mmol/L), which partially recovered over 30 minutes. Preconditioning did not change the [Na(+)](i) rise during ischemia but abolished the large [Na(+)](i) rise on reperfusion, and [Na(+)](i) instead fell (-3.6+/-1.3 mmol/L). In the presence of MIA, the [Na(+)](i) rise was unchanged from ischemia only; on reperfusion, [Na(+)](i) fell (-3.7+/-0.9 mmol/L), similar to the preconditioned hearts. TTX abolished the [Na(+)](i) rise during ischemia (+0.3+/-0.7 mmol/L), and the increase on reperfusion was similar to ischemia only. We conclude that the rise of [Na(+)](i) during ischemia is caused by Na(+) entry through persistent Na(+) channels. The rise of [Na(+)](i) on reperfusion is caused by activation of the Na(+)/H(+) exchanger and is blocked by MIA and by preconditioning. It is known that the Na(+)/H(+) exchanger is inhibited during ischemia; the present result suggests that this inhibition is prolonged into the early part of reperfusion by preconditioning. To test this hypothesis, we measured the time course of pH(i) recovery after ischemia and preconditioning. Preconditioning slowed the rate of pH(i) recovery after ischemia, providing further support for the hypothesis that preconditioning inhibits the Na(+)/H(+) exchanger during early reperfusion. This inhibition of the Na(+)/H(+) exchanger during reperfusion prevents Na(+) entry, and therefore Ca(2+) loading, and is part of the protective pathway involved in preconditioning.