How are myofibrils assembled in skeletal muscles? The current authors present evidence that myofibrils assemble through a three-step model: premyofibrils to nascent myofibrils to mature myofibrils. This three-step sequence was based initially on studies of living and fixed cultured cells from cardiac muscle. Data from avian primary muscle cells and from a transgenic skeletal mouse cell line indicate that a premyofibril model for myofibrillogenesis also holds for skeletal muscle cells. Premyofibrils are characterized by minisarcomeres bounded by Z-bodies composed of the muscle isoform of alpha-actinin. Actin filaments are connected to these Z-bodies and to the mini-A-bands composed of nonmuscle myosin II filaments. Nascent myofibrils are formed when premyofibrils align and are modified by the addition of titin and muscle myosin II filaments. Mature myofibrils result when nonmuscle myosin II is eliminated from the myofibrils and the alpha-actinin rich Z-bodies fuse as the distance between them increases from 0.5 microm in premyofibrils to 2 to 2.5 microm in the mature myofibrils.