The structure and development of the myotome has been extensively studied in birds and amphibians but few studies have systematically addressed its development in mammals. We have used a transgenic mouse carrying an nLacZ marker coupled to a myosin light chain 3F promoter to describe the structure of the developing mammalian myotome. Through studies of transgene expression pattern, coupled with immunohistochemistry for the muscle structural proteins desmin and slow myosin heavy chain we describe a gradient of maturity for the cells within the developing myotome. Our results show that the earliest myocytes of the mammalian myotome span the rostrocaudal extent of the somite and have single large nuclei which localise centrally within the myotome. Throughout the period of study the myotome is more mature ventrally than dorsally and cells comprising the medial aspect of the myotome are younger than those lying laterally. Immunohistochemistry for the earliest expressed muscle regulatory factor (myf-5) is used to define areas of the myotome contributing new myogenic cells. In the early myotome small, round, myf-5-expressing cells are found extensively within the dorsomedial aspect of the dermamyotome and also within the entire rostral and caudal dermamyotomal lips. They subsequently appear within the central zone of the myotome, adjacent to the medially curled rostral and caudal dermamyotomal lips, and there begin to elongate symmetrically. As the myotome enlarges, myf-5 expression is always restricted to the most medial aspect of the myotome, adjacent to the least mature myocytes, marking the site of addition of new myogenic cells. Together, these results allow development of a model of mammalian myotome formation where growth occurs medially by addition of new cells from both rostral and caudal dermamyotome lips, while more mature myocytes are displaced laterally. Furthermore, early myotomal myocytes differentiate in the absence of MyoD expression, unlike later myotomal myocytes. This, along with their distinct morphology, suggests these cells may form a separate lineage of pioneer myogenic cells.