Altered Na⁺/Ca²⁺ exchanger (NCX) protein expression or activity is thought to contribute to various aspects of cardiac pathology. In guinea pig ventricular myocytes, NCX-mediated Ca²⁺ entry is almost entirely responsible for Ca²⁺ overload during hypoxia/reoxygenation. Since Ca²⁺ overload is a common initiator of apoptosis, the purpose of this study was to test the hypotheses that NCX activity is critically involved in initiating apoptosis following hypoxia/reoxygenation, and that hypoxia/reoxygenation-induced apoptosis can be modulated by changes in NCX protein expression or activity. A NCX antisense oligonucleotide was used to reduce NCX protein expression in cultured adult guinea pig ventricular myocytes. Caspase-3 activation and cytochrome C release were used as markers of apoptosis. Hypoxia/reoxygenation-induced apoptosis was significantly decreased in antisense-treated myocytes as compared to untreated control or nonsense-treated myocytes. Pretreatment of cultured myocytes for 24 hours with either endothelin-1 or phenylephrine was found to increase both NCX protein expression and evoked NCX activity, as well as to enhance hypoxia/reoxygenation-induced apoptosis. Control experiments demonstrated that endothelin-1 and phenylephrine did not induce apoptosis on their own, nor did they enhance the apoptotic response in a model of Ca²⁺-dependent, NCXindependent apoptosis. Additional control experiments demonstrated that the NCX antisense oligonucleotide did not alter the apoptotic response of myocytes to either H₂O₂ or isoproterenol. Taken together, these data suggest that the NCX has a critical and specific role in the initiation of apoptosis following hypoxia/reoxygenation in guinea pig myocytes, and that hypoxia/reoxygenation-induced apoptosis is quite sensitive to changes in NCX activity.