Chromosomal protein A24 was first described by its electrophoretic mobility on two-dimensional polyacrylamide gels [I]. It was identified as a conjugated chromatin protein composed of histone 2A [2] and a ubiquitin moiety [3-51 which was linked by an isopeptide bond from a carboxyl terminal glycylglycine to the e-NH2 of lysine 119 of histone 2A [6-81. Protein A24 has been found to be an integral component of a subset of chromatin nucleosome core particles [9-l I]. Levels of protein A24 markedly decreased during nucleolar hypertrophy [12-l 41 and in some active chromatin fractions where free ubiquitin was found [15-l 71. Cleavage of the ubiquitin-histone 2A bond may be involved in some transcription systems [IS]. Moreover, in chromatin the ubiquitin portion of protein A24 turns over throughout interphase [18,19]. Recently, an enzyme, protein A24 lyase, was described which cleaved both free and nucleosomal protein A24 and analysis of the products by gel electrophoresis implied that the enzyme catalyzed a deconjugation [20]. This report is the carboxyl-terminal sequence analysis of the ubiquitin released from purified protein A24 by the lyase. Inasmuch as the lyase released ubiquitin with an intact glycylglycine terminus. The results indicate that the site of cleavage in protein A24 is at the isopeptide linkage between the carboxyl terminal dycine residue 76 of ubiquitin and the e-amino group of lysine residue 119 of histone 2A.