Experiments with purified P-glycoprotein (Pgp) reconstituted into proteoliposomes have conclusively demonstrated that Pgp is an ATP-dependent drug transporter. Detailed biochemical analyses of drug transport by Pgp are beginning to yield a clearer picture of its mechanism. Working with Pgp-rich plasma membrane vesicles from CHRB30 cells, we have recently clarified several aspects of the drug transport mechanism. A major question about drug transport by Pgp is how it can recognize a vast array of unrelated chemical compounds as substrates. The substrate Hoechst 33342 is fluorescent in the lipid bilayer but not in aqueous solution. This property enabled us to show that Pgp transports Hoechst 33342 out of the lipid bilayer, not the aqueous phase. Because Hoechst 33342, like all Pgp substrates, is lipophilic its concentration in the bilayer greatly exceeds its concentration in the aqueous medium. High local substrate concentrations may allow for broad substrate recognition by one or more relatively low affinity binding site(s) within the lipid bilayer. Another fundamental question about Pgp is the number of drug binding sites it possesses. We have found evidence for at least two sites for drug binding and transport that interact in a positively cooperative manner. Initial rates of transport of two Pgp substrates, Hoechst 33342 and Rhodamine 123 by ChRB30 plasma membrane vesicles were measured. Each dye stimulated transport of the other. Additionally, colchicine stimulated Rhodamine 123 transport and inhibited Hoechst 33342 transport. Anthracyclines such as daunorubicin and doxorubicin had the reverse effect. Vinblastine, etoposide, and actinomycin D inhibited transport of both dyes. We interpret these results as follows. One site (R) preferentially recognizes Rhodamine 123, doxorubicin and daunorubicin. The other site (H) preferentially recognizes Hoechst 33342 and colchicine. Vinblastine, actinomycin D, and etoposide interact equally with both sites. Binding of drug at the R site stimulates transport of Hoechst 33342 by the H site and binding of drug at the H site stimulates transport of Rhodamine 123 by the R site. The existence of two drug binding sites on Pgp with different specificities is another way in which Pgp may expand the range of substrates it can transport. A third essential detail of the drug transport mechanism of Pgp is the ratio of substrate molecules transported per ATP hydrolyzed. By comparing the initial rate of Rhodamine 123 transport with the rate of ATP hydrolysis at saturating Rhodamine 123 concentration, we found that, under suitable conditions, Pgp is capable of transporting one Rhodamine 123 molecule per ATP molecule hydrolyzed.