SummaryStudies have been carried out to assess maturation of myofibrillar and mitochondrial proteins in fetal (113 to 140 days gestation), neonatal (30 min to 21 days postpartum), and adult sheep hearts.Ca++‐activated myosin ATPase activity was ˜20% lower in fetal than in adult left ventricular myocardium (1.13 ± 0.06,n= 12,versus1.36 ± 0.07,n= 9, &mgr;moles P1per g protein per sec;P< 0.025). In fetal and neonatal hearts (but not in adult hearts), myosin ATPase activity was slightly higher (˜14%;P< 0.001) in right ventricular tissue than in left ventricular tissue. In contrast to these small changes in myosin ATPase activity, large changes indicative of maturation of energy metabolism occurred in the creatine kinase system: between 115 days gestation and 21 days postpartum, total creating kinase activity increased nearly 8‐fold (0.2 to 1.6 IU/mg cardiac mass), the MM‐creatine kinase isozyme increased 7‐fold (0.2 to 1.5 IU/mg wet weight), and mitochondrial creatine kinase increased more than 25‐fold (< 0.01 to 0.27 IU/mg wet weight). The total creatine pool, but not the ATP pool, increased (from ˜6 to ˜15 nmoles/g tissue). Neither the concentration nor isozyme distribution of lactate dehydrogenase, a glycolytic enzyme, changed during this 7‐wk period of development.SpeculationWe speculate that changes in biochemical composition of energy‐utilizing and ‐producing proteins, coupled with alterations in the structure and cellular distribution of mitochondria and myofibrils, contribute to the changes in mechanical performance characteristic of the maturing heart.