The aim of this study was to quantify the contribution of the Na(+)/H(+) exchanger (NHE) and the Na(+) channel to the rise in cytosolic Na(+) concentration ([Na(+)]) that is seen in anoxic guinea pig ventricular myocytes. [Na(+)] was measured with the use of microfluorometry and was found to rise to 44 mM after prolonged anoxia. This rise was partially sensitive to either TTX or HOE-642, selective inhibitors of the Na(+) channel and NHE1, respectively. [Na(+)] did not significantly rise when both drugs were present, suggesting that other routes of Na(+) entry were insignificant. However, the relative contributions of the NHE and the Na(+) channel were found to be remarkably sensitive to ionic conditions expected to occur during ischemia. The Na(+) channel was the dominant Na(+) source during acidic anoxia. However, the NHE was the dominant Na(+) source during both hyperkalemic anoxia and simulated ischemia (hyperkalemia, low pH, and anoxia). The data suggest that the NHE may prove to be the best pharmacological target to reduce Na(+) entry during true ischemia and that inhibition of Na(+) influx could contribute strongly to the cardioprotective effects of NHE inhibitors.