To analyze the transcriptional regulatory mechanisms of the myoD gene, we generated transgenic mice bearing a lacZ gene driven by a 6-kb 5′-flanking sequence of the mouse myoD gene including a proximal regulatory region (PRR) and a distal regulatory region (DRR), which are sufficient for activation of muscle-specific transcription in vitro. The expression of the reporter lacZ gene was detected from 10.5 days post coitum in the myotomes, consistent with endogenous myoD mRNA. However, in limb buds and branchial arches, the appearance of the lacZ-positive cells was delayed for one day compared with the endogenous myoD mRNA, suggesting the existence of a different control mechanism among muscle cell lineages. Further, a subset of cells in the central nervous system (CNS), where endogenous myoD mRNA was not detected, expressed the transgene transiently. The same 6-kb MyoD-lacZ gene injected into Xenopus embryos was expressed in the myotomes and in CNS cells at the tailbud stage. Deletion analyses in both transgenic mice and Xenopus embryos indicated that the DRR and PRR were together sufficient for the expression pattern in skeletal muscle. In addition, analysis in Xenopus indicated that a third enhancer region between -1.3 kb and -275 bp could substitute for the DRR. The functional conservation of the regulatory region of the mouse myoD gene in Xenopus embryos suggests that the regulatory pathway that activates myoD gene transcription in axial muscles is conserved among vertebrates.