Myogenesis proceeds stepwise from pluripotential stem cell to differentiated myotube. The precise number of transitions that occur along the developmental pathway remains to be determined. We examined the myogenic pathway as modelled by mouse mesodermal stem cell and muscle cell lines for stage-specific alterations in the chromatin structure of the acetylcholine receptor delta and gamma subunit genes. We reasoned that such an analysis would allow us to observe either the primary events in the activation of these muscle-specific genes or processes secondary to the binding of muscle-specific regulatory proteins. We probed chromatin structure with DNase I (deoxyribonuclease I) and precisely mapped to the 5' ends of the delta and gamma genes DNase I hypersensitive (DH) sites whose induction is unique to each myogenic stage. Putative mesodermal stem cells have the simplest pattern of DH sites with no sites near the 5' ends of the delta and gamma genes, whereas differentiated myotubes express the most complex pattern; the myoblast pattern is intermediate and of two types. In muscle cell lines where differentiation must be induced the myoblasts have a simple pattern (one more site than stem cells); in muscle lines where differentiation is spontaneous the myoblasts express a complex pattern of DH sites (one less site than myotubes). Inducible myoblasts seem to be arrested in an earlier step in the myogenic pathway than spontaneously differentiating myoblasts. Thus, myogenic activation of acetylcholine receptor subunit genes appears to be a stepwise process that can be detected by chromatin structural changes specific to four distinct stages of muscle development: stem cell, early myoblast, late myoblast, and differentiated myotube.