P-glycoprotein (Pgp) is a drug-translocating ATPase responsible for multidrug resistance in cancer. Although it is well-established that Pgp exhibits drug-dependent ATPase and ATP-dependent drug transport functions, the mechanism by which these two reactions are coupled remains unclear. We have shown recently that proteolytic cleavage of the linker region, which joins the NH2 and COOH halves of the Pgp molecule, results in a Pgp form that exhibits drug-independent and -dependent ATPase activities (Nuti et al., (2000) Biochemistry 39, 3424-3432; Nuti, S. L., and Rao, U. S. (2002) J. Biol. Chem. 277, 29417-29423). To understand the mechanism underlying this phenomenon, we used the procedure of vanadate-mediated trapping of the Pgp transport cycle intermediates to determine the steps in the catalytic cycle that are being regulated by the linker region. We show that vanadate stably traps Pgp under two different conditions, one in the presence of ATP alone and the other in the presence of ATP and drug, suggesting the existence of two Pgp conformations. These two conformations, one mediating basal and the other drug-stimulated ATPase reactions, represent different transport cycle intermediates of Pgp, because arresting Pgp in either conformation prevents the catalytic cycle from proceeding to completion. The results also show that these two conformations are uncoupled and appear simultaneously in Pgp that was cleaved in the linker region. These results together suggest that Pgp assumes at least two distinct conformational states, which catalyze two ATP hydrolysis events in the drug transport cycle, and the linker region mediates the transition between these two states of Pgp.