During early embryonic development in the frog Xenopus laevis, several muscle-specific actin genes encoding distinct actin protein isoforms are activated in cells of the embryonic muscle. In addition to the cardiac (or alpha 1) and skeletal (or alpha 2) actin genes, a third muscle-specific actin gene is expressed in the same embryonic tissue. We have determined the complete nucleotide sequence of this third gene and examined its expression in embryonic and adult tissues. During embryogenesis, this femoral (alpha 3) actin gene is activated several hours later than its cardiac and skeletal counterparts and its transcripts are first detected after neurulation. The gene encodes a skeletal-type actin protein and is expressed exclusively in skeletal muscle in the adult frog. Two copies of this gene have been isolated from the tetraploid species Xenopus laevis, differing by only a few nucleotides in their protein-coding sequence. The related, diploid species, Xenopus tropicalis, possesses a single copy of the alpha 3 gene and its transcript is similarly conserved in nucleotide sequence. However, the X. tropicalis gene is expressed exclusively in embryonic stages of development. Comparison of the X. laevis and X. tropicalis alpha 3 gene promoters reveals extensive sequence homology, including several copies of a repeated motif that is common to other vertebrate striated-muscle actin gene promoters.