The functional development of the creatine kinase system has been studied in rabbit heart during perinatal growth. Fiber bundles were obtained from left ventricles of fetal rabbits at the 30th day of gestation, newborn rabbits aged 1, 3, 8, and 17 days, and adult rabbits. Total creatine kinase activity was constant during perinatal development, whereas myofibrillar bound creatine kinase activity increased 15-fold during the first postnatal week. Functional activity of myofibrillar creatine kinase was assayed in Triton X-100-skinned fibers by its ability to induce active tension in the absence of ATP or to relax rigor tension. It was very low in 1-day-old newborns and increased during the first 2 weeks to reach adult levels 17 days after birth. Functional activity of mitochondrial creatine kinase was determined in saponin-skinned fibers. Creatine-stimulated respiration appeared only after birth and increased gradually between 1 and 17 days after birth. The results show that, although the two creatine kinase isoforms (mitochondrial and myofibrillar) are expressed at different stages during development, their functional activities appear in parallel in mitochondria and myofibrils. Early postnatal development is characterized by binding of creatine kinase isoenzymes to intracellular organelles. Such compartmentation participates in the postnatal cardiac cellular maturation.