The electrophoretic mobility of the cardiac Na(+)-Ca(2+) exchange protein is different under reducing and nonreducing conditions. This mobility shift is eliminated in a cysteine-less exchanger, suggesting that the presence or absence of an intramolecular disulfide bond alters the conformation and mobility of the exchanger. Using cysteine mutagenesis and biochemical analysis, we have identified the cysteine residues involved in the disulfide bond. Cysteine 792 in loop h of the exchanger forms a disulfide bond with either cysteine 14 or 20 near the NH(2) terminus. Because the NH(2) terminus is extracellular, the data establish that loop h must also be extracellular. A rearrangement of disulfide bonds has previously been implicated in the stimulation of exchange activity by combinations of reducing and oxidizing agents. We have investigated the role of cysteines in the stimulation of the exchanger by the combination of FeSO(4) and dithiothreitol (Fe-DTT). Using the giant excised patch technique, we find that stimulation of the wild type exchanger by Fe-DTT is primarily due to the removal of a Na(+)-dependent inactivation process. Analysis of mutated exchangers, however, indicates that cysteines are not responsible for stimulation of the exchange activity by Fe-DTT. Ca(2+) blocks modification of the exchanger by Fe-DTT. Disulfide bonds are not involved in redox stimulation of the exchanger, and the modification reaction is unknown. Modulation of Na(+)-dependent inactivation may be a general mechanism for regulation of Na(+)-Ca(2+) exchange activity and may have physiological significance.