The heat-stable polypeptide of the ATP-dependent proteolytic system was previously found to form covalent conjugates with proteins and to be activated by ATP in an adenylylation mechanism. To identify the functional amino acid of the polypeptide, the activated residue was specifically labeled by the reductive cleavage of the intermediate with [3H]borohydride. Following acid hydrolysis, the reduced labeled derivative was found to be completely oxidizable by periodate with formation of [3H]formaldehyde, and was identified as ethanolamine by thin layer chromatography, electrophoresis, and amino acid analyzer chromatography. These results indicate that the activated amino acid residue of the polypeptide is COOH-terminal glycine.