Abstract Argininosuccinase from bovine liver with a molecular weight of 202,000 completely dissociates at pH 9 or 10 to 5.6 S subunits. The catalytically active tetramer can be reconstituted by bringing the pH back to 7.5. Determined by sedimentation equilibrium the subunit molecular weight is 100,000 ± 2,500, corresponding to that of a dimer. Sedimentation velocity experiments show that initially the dissociated preparation consists of primarily dimer. Polymerization, apparently through the participation of dimers, is observed after long periods as sedimentation equilibrium analyses show the presence of small amounts of tetramer not in chemical equilibrium with the dimer, and some hexamer. In concentrated solutions of urea or guanidine hydrochloride, the molecular weight by sedimentation equilibrium is 50,400 ± 1,100. A similar value was estimated by gel electrophoresis in sodium dodecyl sulfate in the presence or absence of reducing agent. The four polypeptide chains are, therefore, identical in size. The enzyme is devoid of disulfide bonds; in guanidine hydrochloride 16 moles of free sulfhydryl groups were determined with 5,5'-dithiobis(2-nitrobenzoate), and this value is equal to the number of half-cystine residues per mole of protein. End group analyses showed that the carboxyl-terminal amino acid is glutamine, the penultimate residue is leucine, and there are four identical terminal sequences per mole. Cyanogen bromide cleavage produced 13 to 14 peptides, one-fourth of the expected number of fragments based on a methionine content of 51 residues per mole of protein. In accord with these results, tryptic peptide patterns showed only one-fourth of the total number of peptides predicted from the lysine and arginine content of the enzyme. The number of peptides containing several other specific amino acid residues was also consistent with the number predicted from an average distribution of residues among four polypeptide chains. These data show that argininosuccinase is composed of four subunit polypeptide chains that are apparently identical, or very similar, in amino acid sequence. The distribution of the 16 sulfhydryl groups was related to quaternary structure from studies with sulfhydryl reagents. In the active tetramer, four sulfhydryl groups are readily accessible; presumably, they are located on or near the surface of the enzyme and are remote from the active sites, as disulfide or mercaptide formation with sulfhydryl reagents does not affect catalytic activity. A second group of four is masked within the regions of bonding between dimeric subunits; they are released concomitantly with dissociation of the tetramer to dimers. The remaining eight sulfhydryl groups only become accessible as the dissociation of dimers to monomers proceeds in the presence of strong dissociating agents. From the results of dissociation behavior and sulfhydryl reactivity, at least two modes of subunit interactions can be distinguished. Electron micrographs of negatively stained argininosuccinase support this four-subunit model. The model is discussed with respect to modes of dissociation, geometric arrangement, and symmetry.