Preassay-incubation of the highly purified human erythrocyte adenine phosphoribosyltransferase (EC 2.4.2.7) (AMP pyrophosphorylase) with one of its substrates, 5-phosphoribosyl 1-pyrophosphate (PRibPP), changes the apparent V max value of the enzyme reaction. The extent of inhibition by preassay-incubation with an inhibitor, fructose 1,6-diphosphate (FDP), or a destabilizer, hypoxanthine (Hx), is found not to be proportional to the amount of the inhibitor present. The maximum inhibition achieved by preassay-incubation was about 40%. The PRibPP, FDP, and Hx induced changes in AMP pyrophosphorylase do not require the presence of divalent ions. The inhibtion of AMP pyrophosphorylase produced by preincubation with Hx was prevented when PRibPP was added to the preassay-incubation system. However, the preassay-incubation effect of FDP was only partially diminished under the same conditions. Contrary to the PRibPP-bound AMP pyrophosphorylase, the adenine-bound enzyme was found to be more heat labile than the unbound enzyme. Similar thermal instability was also observed with FDP- and Hx-bound enzyme. Our experimental results indicate that a conformational change of AMP pyrophosphorylase induced by the binding of metabolites is a slow process as compared to the overall catalytic reaction. This hysteretic characteristic of AMP pyrophosphorylase may be one of the regulatory mechanisms in purine intermediary metabolism.