Adenylosuccinate synthetase (AS-synthetase) was purified from the yeast Saccharomyces cerevisiae. The purification procedure included chromatography on DEAE-cellulose, phosphocellulose, and heparin-agarose. The pH and temperature optima for the enzyme activity (7.0 and 35 degreesC, respectively) and also pH and thermostability of AS-synthetase were determined. The native form of the enzyme exists as a dimer. The Km values for IMP, GTP, and L-aspartate are 1.7, 0.16, and 6.7 mM, respectively. ATP cannot be used instead of substrate GTP, whereas 2'-dGTP and dd-GTP are able to substitute for GTP in the reaction. ITP also can be a substrate as an analog of GTP and as an analog of IMP. Two intermediates of purine nucleotide biosynthesis de novo, 5-amino-4-(N-succinocarboxamide)imidazole ribonucleotide (ASCIR) and 5-amino-4-carbamoyl-imidazole ribonucleotide (ACIR), inhibit AS-synthetase. Hydroxylamine and aspartate analogs also inhibit the enzyme. Effective binding requires a four-carbon-atom chain and unsubstituted amino group; the charge of the beta-carboxy group is not necessary. Comparison of primary structures and substrate specificity of yeast ASCIR- and AS-synthetases suggests independent origin of these proteins.