Resistance to multiple chemotherapeutic drugs is a major obstacle to the success of cancer treatment. P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily, is a major cause of multidrug resistance. Pgp effluxes a broad range of hydrophobic compounds from cells driven by ATP hydrolysis at two cytosolic nucleotide binding (NB) domains. Pgp was converted to the vanadate-trapped transition state complex Pgp·ADP·V i, and the fluorescent probe 2-(4'-maleimidyl-anilino)naphthalene-6-sulfonic acid (MIANS) was used to label the Walker A Cys residue at the vacant NB site. After release of trapped ADP, the second NB site was labeled with another thio-reactive fluorescent probe, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). MIANS fluorescence emission in double-labeled Pgp was highly quenched as a result of fluorescence resonance energy transfer (FRET) to NBD. The estimated distance between the two fluorophores was ~16 A? and ~22 A? for Pgp labeled in the membrane or in detergent solution, respectively, suggesting that the two catalytic sites are relatively close to each other, as in the X-ray crystal structure of Rad50cd and BtuCD. The fluorescent dye Hoechst 33342 (H33342) binds to Pgp with high affinity. Pgp was labeled with NBD at both NB sites in the resting state, or at one NB site in the transition state. H33342 fluorescence was quenched when bound to NBD-labeled Pgp, and FRET measurements estimated the distance separating bound H33342 from the NBD group to be ~38 A? and ~34 A? in the resting state and the transition state, respectively. This places the H33342 binding site 10-14 A? below the membrane surface, within the cytoplasmic leaflet. Fluorescence quenching of transition state MIANS-Pgp indicated that the empty NB site can bind nucleotides, with similar affinity to the resting state. Drug binding increased the affinity of resting state Pgp for nucleotide by up to ~4-fold, while drug binding decreased the affinity of transition state Pgp by up to ~3-fold. Fluorescence quenching of MIANS and Trp residues indicated that transition state Pgp had only a ~2-fold lower affinity for drug binding. These results suggest that drug has already been transported by Pgp before the transition state complex is formed.