Cardiac muscle fibers in rats at 1 and 5 days after birth showed little evidence of M-bands. An ultrastructural analysis of myofibrils failed to demonstrate dense M-band material in longitudinal sections or M-bridges in transverse sections of sarcomeres. M-bands began to increase in number after 5 days of postnatal life and were present in 60% of all sarcomeres at 11 days of age. Polypeptides with molecular weights of 190,000 (Ma) and 175,000 (Mb) were obtained by polyacrylamide-sodium dodecyl sulfate (SDS) gel electrophoresis of myofibril preparations. These two proteins were found in both 1- and 11-day-old rats and were considered to be specific components of the M-band. The densitometric analysis demonstrated that Ma and mb polypeptides increased approximately 2-fold during the interval from 1 to 11 days after birth. These structural and biochemical changes of the M-band material in myocytes appear to be related to the maturation of contractile function in the young heart.