To identify regulatory events occurring during myogenesis, we characterized the transcriptional regulation of a Drosophila melanogaster actin gene, Actin 57B. Act57B transcription is first detected in visceral muscle precursors and is detectable in all embryonic muscles by the end of embryogenesis. Through deletion analysis we identified a 595 bp promoter element that was sufficient for high levels of expression in all three muscle lineages. This fragment contained a MEF2 binding site conserved between D. melanogaster and Drosophila virilis which bound MEF2 protein in embryo nuclear extracts. Mutation of the MEF2 site severely reduced promoter activity in embryos, and in Mef2 mutants Act57B expression was severely decreased, demonstrating MEF2 is an essential regulator of Act57B. We also showed that MEF2 likely acts synergistically with factors bound to additional sequences within the 595 bp element. These findings underline the importance of MEF2 in controlling differentiation in all muscle lineages. Our experiments reveal a novel regulatory mechanism for a structural gene where high levels of expression in all embryonic muscles is regulated through a single transcription factor binding site.