In an attempt to clarify the regulatory mechanism that accounts for the shift of protein A24 in the mitotic cycle, we demonstrated the existence of an enzyme, provisionally termed isopeptidase, that cleaves A24 stoichiometrically into histone H2A and ubiquitin. Properties of this enzyme are (i) most eukaryotes, including mammals, amphibia, chicken, and yeast, contain isopeptidase in the cytoplasm; (ii) a significant increase in enzyme binding to chromatin occurs when cells enter mitosis; (iii) Escherichia coli does not contain isopeptidase; (iv) isopeptidase has a molecular weight of 38,000; (v) at an ionic strength that induces globular conformation of H2A, isopeptidase activity is repressed; (vi) a SH group is an essential cofactor; and (vii) most divalent cations (except Mg2+ and Ca2+) are inhibitory. In view of the stoichiometric conversion of A24 into H2A and ubiquitin by isopeptidase in vitro, A24 probably contains a Gly-Gly dipeptide in isopeptide linkage but no other intervening polypeptides. Since ubiquitin in various eukaryotes binds to protein other than H2A, and is proteolytically released, isopeptidase probably acts on isopeptide bonds in general and not uniquely on those of A24. Inasmuch as isopeptidase is present throughout the cell cycle, the level of A24 in chromatin appears to be controlled by a balance between isopeptidase and an as yet unestablished H2A-ubiquitin ligase.