IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, a promising target for cancer chemotherapy, was purified about 5,000-fold to homogeneity from rat transplantable hepatoma 3924A. Sephacryl S-400 gel filtration showed that the molecular weight of the native enzyme protein was about 245,000. SDS electrophoresis indicated that the molecular weight of the subunits was about 60,000. Thus, the enzyme has a tetrameric structure. The double reciprocal plotp for substrate dependence yielded Km values for IMP and NAD+ of 23 and 65 uM, respectively. Excess NAD+ caused substrate inhibition in the purified enzyme. The enzyme requires potassium ions; an apparent Km value for K+ was 7.8 mM. Product-inhibition studies showed that XMP was competitive with IMP (Ki = 136 uM) and noncompetitive with NAD+. NADH exerted uncompetitive inhibition with respect to IMP (Ki = 210 uM) and mixed type with NAD+ (Ki(slope) = 290 uM). This inhibitory pattern suggests an ordered Bi-Bi mechanism for the enzyme reaction in which IMP binds to the enzyme first, followed by NAD+. NADH dissociates from the ternary complex after rearrangement, and finally XMP is released. XMP interacts with the free enzyme and competes for the ligand site with IMP, while NADH binds to the enzyme XMP complex. (Supported by Outstanding Investigator Grant CA-42510 to G.W.)