Adenylosuccinate synthetase (IMP:L-aspartate ligase (GDP-forming), EC 6.3.4.4) was purified about 750-fold to a homogeneous state from Yoshida sarcoma ascites tumor cells. A yield of 38% purified enzyme was achieved by a procedure including affinity chromatography on hadacidin-Sepharose 4B. Ultracentrifugal analyses showed that the molecular weight of the native enzyme was 102 000 with an s20,w value of 4.5 and that the molecular weight in 6 M guanidine-HCl was 47 000. These values indicate that the native enzyme is composed of two subunits. The isoelectric point was determined to be 5.9 by isoelectric focusing. The optimum pH for activity was 6.8-7.0. The Km values for IMP, aspartate and GTP were calculated to be 4.1, 9.8 and 0.7 . 10(-4) M, respectively. The antibiotic, hadacidin was strongly inhibitory, causing competitive inhibition with respect to aspartate with a Ki value of 2.5 . 10(-6) M. Nucleoside mono- and diphosphate also inhibited the enzyme activity, but their inhibitions were not apparently specific. The purified enzyme showed full activity in the presence of Mg2+, and Mg2+ could be partially replaced by Mn2+, Co2+, Ca2+ or Cu2+. Divalent metal ions, such as Cd2+, Pb2+, Zn2+, Cu2+ and Mn2+, interfered with the activity by antagonizing Mg2+. Hg2+ or PCMB inactivated the enzyme, suggesting that an SH-group may be important for activity.