For characterization of the growth pattern of cardiac myocytes during posthatching development, cardiac myocytes were enzymatically isolated from the ventricles of 1-, 15-, 29-, and 42-day-old chickens for measurement of myocyte nucleation, length, width, volume, and number, and for immunolabeling of cytoskeletal proteins. Ventricular myocyte number increased 156% from day 1 to day 42. Average cell volume increased more than 400%, and myocytes lengthened 125%, but cell width only increased 53% during this period. All myocytes were mononucleated at day 1. At day 15, 18% of myocytes became binucleated with < 1% of myocytes containing more than two nuclei. Interestingly, binucleated myocytes were able to divide with two nuclei going through mitosis at the same time. As demonstrated by staining with tubulin and alpha-actinin antibodies, two mitotic spindles and two cleavage furrows were formed in dividing binucleated myocytes. At day 42, binucleated myocytes increased to 44% with 11% of myocytes containing more than two nuclei. Sarcomeric alpha-actinin was partially disassembled in prometaphase and was reorganized into regular Z lines of sarcomeres in telophase. Desmin was disassembled in prophase and was reassembled during late telophase. These results suggest that chicken myocytes undergo hypertrophy and continue to proliferate during posthatching maturation, although it is currently believed that myocytes of all vertebrates withdraw from the cell cycle shortly after birth. We provide direct evidence for the first time of in vivo myocyte division in 6-wk-old chicken hearts.