IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD to NADH. This reaction is the rate-limiting step of guanine nucleotide biosynthesis. IMPDH is a target of immunosuppressive, antiviral, anticancer, and antiparasitic chemotherapy. We have determined a minimal kinetic mechanism for human IMPDH type II using NAD analogs, isotope effects, hydride exchange, and presteady state kinetics. The values of kcat for the NAD analogs are similar despite a great variation in the structure and reactivity of the compounds. This observation suggests that a common step is rate-limiting, i.e., either hydrolysis of the E-XMP* intermediate or release of XMP. No Vm isotope effect is observed when 2-2H-IMP is the substrate, which indicates that hydride transfer is fast. This conclusion is confirmed by the observation of a burst of NADH production under presteady state conditions. These observations further suggest that either E-XMP* hydrolysis or XMP release is rate-limiting. V/Km deuterium isotope effects are observed for both substrates (1.9 for IMP and 2.5 for NAD), which indicates that substrate association is random. This result contradicts previous conclusions based on product inhibition studies. No NADH consumption is observed in the presence of XMP and IMPDH, which indicates that the overall reaction is irreversible. NADH consumption is observed in the presence of thio-NAD, IMP, and enzyme. These observations indicate that NADH traps the E-XMP* intermediate and demonstrates that hydride transfer is reversible. At infinite NADH, all of E-XMP* is trapped by NADH, as indicated by the equivalence of the rates of consumption of thio-NAD and NADH. Therefore the release of products is ordered, with NADH release preceding hydrolysis of E-XMP*.