Monoclonal antibodies specific to the light- and heavy-chain subunits of chicken skeletal muscle myosin have been used to identify fast and slow myosin-containing fibers in the thigh muscles of embryonic and adult chickens and to determine when in development diversification of muscle fiber types first occurs. Primary generation fibers which expressed different MLC and MHC types were evident within the dorsal and ventral premuscle masses and in the first muscles to form in the limb. These early embryonic muscle fiber types became distributed among and within the individual muscles of the thigh in a characteristic spatial pattern which served as a "blueprint" for guiding future muscle development and predicting the future fiber composition of the muscle. Despite the continuous addition of muscle fibers to the limb throughout development, the pattern remained unchanged. Neither the time of appearance, initial specialization, nor characteristic distribution of these primary fiber types within the limb was altered during the early embryonic period by chronic neuromuscular paralysis induced by D-tubocurarine. In contrast, muscles at later stages of embryonic development were markedly affected by such treatments and underwent atrophy and loss of differential staining characteristics. These results demonstrate that diversification of fibers in terms of myosin content is one of the earliest events in the formation of these muscles and suggest that the development of avian muscles be divided into two phases: an embryonic phase during which fibers of differing myosin content appear independently of innervation to become distributed in a specific topographic pattern within each muscle as it forms, followed by a fetal phase during which innervation becomes essential for maintaining this pattern and modulating the myosin content of its fibers.