Two succinyl-CoA synthetases, one highly specific for GTP/GDP and the other for ATP/ADP, have been purified to homogeneity from pigeon liver and breast muscle. The two enzymes are differentially distributed in pigeon, with only the GTP-specific enzyme detected in liver and the ATP-specific enzyme in breast muscle. Based on assays in the direction of CoA formation, the ratios of GTP-specific to ATP-specific activities in kidney, brain, and heart are approximately 7, 1, and 0.1, respectively. Both enzymes have the characteristic alpha- and beta-subunits found in other succinyl-CoA synthetases. Studies of the alpha-subunit by electrophoresis, mass spectrometry, reversed-phase high performance liquid chromatography, and peptide mapping showed that it was the same in the two enzymes. Characterization of the beta-subunits by the same methods indicated that they were different, with the tryptic peptide maps providing evidence that the beta-subunits likely differ along their entire sequences. Because the two succinyl-CoA synthetases incorporate the same alpha-subunit, the determinants of nucleotide specificity must reside within the beta-subunit. Determination of the apparent Michaelis constants showed that the affinity of the GTP-specific enzyme for GDP is greater than that of the ATP-specific enzyme for ADP (7 versus 250 microM). Rather large differences in apparent Km values were also observed for succinate and phosphate.