The gene for aldolase A in mouse has been shown to be regulated by alternative promoters with attendant alternative first exons. The distal promoter/exon M functions only in muscle while the proximal promoter/exon H is active in early muscle development and in most other tissues. We have analyzed the developmental expression of M and H promoters in mouse throughout myogenesis both in vitro and in vivo. In C2C12 cells RNase protection assays revealed the M promoter is induced within 24 hr of the onset of myogenic differentiation, and both M- and H-specific mRNAs accumulate over 5 days in culture. Nuclear run-on transcription and in situ hybridization with an exon-specific probe demonstrate that the H promoter remains transcriptionally active even in differentiated myotubes. The in vitro results were then compared to similar RNase protection studies of M and H expression during muscle development in vivo. These data show a marked similarity between promoter activation and steady-state transcript accumulation in vivo and in vitro, but within a limited developmental time frame (E15 to 1 week postnatal). In situ hybridizations suggest that simultaneous transcription from both promoters may also occur early in muscle development. Furthermore, the M promoter shows no fiber-type restriction until 1 to 3 weeks postnatally, coincident with muscle maturation, while the H promoter remains transcriptionally active at all stages of development and in all fiber types.