The HrMA4 alpha gene for an embryonic muscle actin of the ascidian Halocynthia roretzi is activated at the gastrula stage in differentiating muscle cells exclusively. The 5' upstream region close to the transcription start site of HrMA4 alpha contains several consensus sequences, which include a TATA box at -30, and E-box at -71, a CArG box at -116, and a cluster of three E-boxes between -150 and -190. When deletion constructs of this region, fused with the bacterial gene for beta-galactosidase (lac-Z), were microinjected into fertilized eggs, the reporter gene was expressed in muscle cells of tailbud embryos. Analyses of the deletion constructs suggested that the 103-bp upstream region is sufficient for the appropriate expression of the gene. However, beta-gal activity was very rarely detectable in the case of 82-bp upstream region and no activity was detected in the case of 72-bp upstream region. Mutations in the proximal E-box sequence did not disturb the muscle-specific expression of the reporter gene. These results suggest that rather short sequences between nucleotides -103 and -72 from the transcription start site are associated with the specific expression of HrMA4 alpha.