The reactivity of the ATP-dependent multidrug transporter P-glycoprotein (Pgp) with the conformation-sensitive monoclonal antibody UIC2 is increased in the presence of Pgp transport substrates, ATP-depleting agents, or mutations that reduce the level of nucleotide binding by Pgp. We have investigated the effects of nucleotides and vinblastine, a Pgp transport substrate, on the UIC2 reactivity of Pgp in cells permeabilized by Staphylococcus aureus α-toxin. ATP, ADP, and nonhydrolyzable ATP analogues decreased the UIC2 reactivity; this effect was potentiated by vanadate, a nucleotide-trapping agent. The Hill number for the nucleotide-induced conformational transition was 2 for ATP and ADP but 1 for nonhydrolyzable ATP analogues. The Hill numbers for ATP and ADP were decreased to 1 by mutations in one of the two nucleotide binding sites of Pgp, whereas mutation of both sites greatly diminished the overall effect of nucleotides. Vinblastine reversed the decrease in the UIC2 reactivity brought about by all the nucleotides, including nonhydrolyzable analogues; this effect of vinblastine was blocked by vanadate. These data indicate that UIC2-detectable conformational changes of Pgp are driven by binding and debinding of nucleotides, that nucleotide hydrolysis affects the Hill number for its Pgp interactions, and that Pgp transport substrates promote nucleotide dissociation from Pgp. These findings are consistent with a conventional E1/E2 model that explains conformational transitions of a transporter protein through a series of linked equilibria.