Phosphoglucomutase is inhibited by a complex formed from alpha-D-glucose 1-phosphate (Glc-1-P) and inorganic vanadate (Vi). Both the inhibition at steady state and the rate of approach to steady state are dependent on the concentrations of both Glc-1-P and Vi. Inhibition is competitive versus alpha-D-glucose 1,6-bisphosphate (Glc-P2) and is ascribed to binding of the 6-vanadate ester of Glc-1-P (V-6-Glc-1-P) to the dephospho form of phosphoglucomutase (E). The inhibition constant for V-6-Glc-1-P at pH 7.4 was determined from steady-state kinetic measurements to be 2 x 10(-12) M. The first-order rate constant for approach to steady state increases hyperbolically with inhibitor concentration. The results are consistent with rapid equilibrium binding of V-6-Glc-1-P to E, with dissociation constant 1 x 10(-9) M, followed by rate-limiting conversion of the E.V-6-Glc-1-P complex to another species, E*.V-6-Glc-1-P, with first-order rate constant 4 x 10(-2)s-1. The rate constant determined for the reverse reaction, conversion of E*.V-6-Glc-1-P to E.V-6-Glc-1-P, is 2.5 x 10(-4)s-1. Formation of E*.V-6-Glc-1-P can also occur via binding of glucose 6-vanadate to the phospho form of phosphoglucomutase (E-P) followed by phosphoryl transfer and rearrangement of the enzyme-product complex.