Age dependent differences in mechanical performance and morphometric and electron microscopic characteristics of atrial and ventricular trabeculae are described. At 3 months, atrial and ventricular trabeculae develop the same amount of force. At 9 months, the ventricular muscle develops twice as much force as its atrial counterpart, although shortening is almost identical in both muscles. At any age, velocity of shortening of atrial trabeculae is at least twice that of the ventricular muscles. Stereological data indicate that atrial and ventricular working myocytes maintain fixed volume fractions of myofibrils (70%) and mitochondria (25%) between 3 and 9 months of age. A broad frequency distribution of sarcomere length was measured at L(max) in muscles of the younger age groups. More than 80% of sarcomeres of adult atrial and ventricular myocytes clustered around 2.05 to 2.25 μm; only 30% of sarcomeres of younger atrial myocytes and 45% of sarcomeres of younger ventricular myocytes were within that length bracket. About 45% of sarcomeres in younger atrial muscles had lengths in excess of 2.35 μm; less than 3% of sarcomeres were longer than 2.35 μm in adult atrial muscles. Sarcomere lengths cluster more and more around the mean with increasing age, suggesting that with maturation there is a more homogeneous recruitment of sarcomeres. At both ages, there is a marked difference between atrial and ventricular myocytes whether examined in terms of morphological development or functional performance. We conclude that any work correlating myocardial structure and function must account for two things: the site from which the muscle was excised and the age of the donor heart.