Objectives: Apoptosis is a mechanism for deletion of injured or obsolete cells that is distinct from necrosis and mediated by mitochondrial release of cytochrome c caspase activation. Because myocardial apoptosis is a part of normal fetal and postnatal maturation, we hypothesize that neonatal myocardium is more vulnerable to undergo myocardial apoptosis than mature myocardium after cardioplegic arrest. Methods: Newborn and mature lambs (n = 5 in each group) underwent cardiopulmonary bypass, antegrade crystalloid hyperkalemic cardioplegic arrest for 60 minutes, and a 6-hour recovery period. Myocardium was examined by using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling (TUNEL), Western blotting, in vitro kinase assays, and fluorometric assays of the activity of caspases 3, 8, and 9. Myocardium from nonoperated control subjects (n = 5 in each age group) was also obtained. Results: More TUNEL-positive nuclei were present in the newborn postcardioplegic myocardium (P = .04). Caspase 3, 8, and 9 activities were 1.6-fold, 1.5-fold, and 1.4-fold greater in the newborn postcardioplegic myocardium (P = .04, P = .01, and P = .01, respectively). The Bax/Bcl-2 ratio was higher in the newborn postcardioplegic myocardium (P = .04). Apoptosis signal-regulating kinase 1 activity and cleaved caspase 3 levels were higher in the newborn postcardioplegic myocardium (P = .02 and P = .009). Mitochondrial release of cytochrome c was greater in the newborn postcardioplegic myocardium (P = .009). Conclusions: The increased Bax/Bcl-2 ratio in the newborn myocardium suggests a proapoptotic state that is manifested by greater TUNEL staining, cytochrome c release, and cleavage of caspase 3. Increased apoptosis signal-regulating kinase 1 activity suggests greater oxidative stress, immature mechanisms to ameliorate oxidative stress, or both in the neonatal myocardium. Mitochondrial release of cytochrome c suggests that apoptosis-related mitochondrial dysfunction might contribute to early postoperative myocardial dysfunction in the neonate.