P-glycoprotein (Pgp) has been associated with multidrug resistance as a major impediment to successful chemotherapy by excluding many structurally diverse therapeutic agents from cancer cells. Details regarding substrate binding and trajectory as well as the molecular basis by which ATP hydrolysis is coupled remains to be elucidated. Tetramethylrosamine (TMR) analogues were prepared with the intent to characterize the "R" binding site and their K_M^D and V_M^ATP values were determined using purified Cys-less Pgp. Substitution of heavy-chalcogen atoms for oxygen in the xanthylium core resulted in negligible changes in ATPase activity, however, substitutions at the 9-position with substituents smaller than the napthyl that vary in number, position and flexibility of the hydrogen-bond acceptors largely influence Pgp activity. Between library members of differing TMR scaffolds, a 1000-fold variation in ATPase activity was observed. These results establish predictions valuable for the design of tighter binding analogues useful for the inhibition of Pgp.