The ABC multidrug transporter P-glycoprotein (Pgp, ABCB1) can transport structurally diverse substrates from the lipid bilayer. Pgp binds its substrates inside a large pocket with multiple sub-sites. In the present work, a thiol-reactive agent, (2-pyridyl)dithiobimane, was covalently linked to 5 of the 7 Cys residues in hamster Pgp, with substantial ATPase activity retained. Three of the labelled Cys residues are located in the transmembrane domain, close to the substrate-binding pocket. Binding affinity determination using Trp and bimane fluorescence for tetramethylrosamine (R-site), Hoechst 33342 (H-site) and 6-(dimethylamino)-2-[4-[4-(dimethylamino)phenyl]-1,3-butadienyl]-1-ethyl perchlorate (both sites), showed similar Kd values for native Pgp and Pgp-bimane adducts, suggesting that the binding pocket can accommodate more than one substrate simultaneously. Reconstituted Pgp-bimane proteoliposomes showed the ability to transport all three substrates across the membrane, although with lower Vmax values compared to native Pgp. Thus, the Pgp-bimane adduct can transport substrates when more than one substrate occupies the binding pocket.