Highly ATP- and GTP-specific isoforms of succinyl-CoA synthetase in pigeon incorporate the same alpha-subunit, but different beta-subunits (Johnson, J. D., Muhonen, W. W., and Lambeth, D. O. (1998) J. Biol. Chem. 273, 27573-27579). The sequences of the mature subunits were determined by methods based on reverse transcription-polymerase chain reaction. The 306-residue mature alpha-subunit in pigeon shows >88% identity to its homologues in pig and rat. The sequences of the mature ATP- and GTP-specific beta-subunits (A-beta and G-beta, respectively) in pigeon are 54% identical. These sequences were used to identify expressed sequence tags for human and mouse that were highly homologous to G-beta and A-beta, respectively. The sequences for mature A-beta and G-beta in mouse and human were completed and verified by polymerase chain reaction. The sequence of A-beta in pig was also obtained. The mammalian A-beta sequences show >89% identity to each other; the G-beta sequences are similarly related. However, pairwise comparisons of the A-beta and G-beta sequences revealed <53% identity. Alignment with two sequences of the beta-subunit in Caenorhabditis elegans suggests that the A-beta and G-beta genes arose by duplication early in the evolution of multicellular eucaryotes. The expression of A-beta is strong in numerous mouse and human tissues, which suggests that ATP-specific succinyl-CoA synthetase also plays an important role in species throughout the animal kingdom.