Overexpression of ATP-dependent drug efflux pumps, P-glycoprotein (Pgp) or multidrug resistance-associated protein (MRP), confers multidrug resistance to tumor cells. Modulators of multidrug resistance block the action of these pumps, thereby sensitizing cells to oncolytics. A potent Pgp modulator is LY335979, which fully sensitizes Pgp-expressing cells at 0.1 μM in cytotoxicity assays and for which Pgp has an affinity of 59 nM. The present study examines its effect on MRP1-mediated drug resistance and cytochrome P-450 (CYP) activity and its ability to serve as a Pgp substrate. Drug resistance was examined with HL60/ADR and MRP1-transfected HeLa-T5 cells. Drug cytotoxicity was unaffected by 1 μM LY335979; leukotriene C4 uptake into HeLa-T5 membrane vesicles was unaffected. Because the substrate specificity of Pgp and CYP3A overlap, the effect of LY335979 on the 1′-hydroxylation of midazolam by CYP3A in human liver microsomes was examined. The apparent K _i was 3.8 μM, ∼60-fold higher than the affinity of Pgp for LY335979. The modulator’s effect on Pgp was evaluated with Pgp-overexpressing CEM/vinblastine (VLB)₁₀₀ and parental CCRF-CEM cells. Both cell lines accumulated [³H]LY335979 equally well and did not efflux [³H]LY335979 during a 3-h incubation, indicating that it is not a substrate of Pgp. Equilibrium-binding studies with CEM/VLB₁₀₀ plasma membranes and [³H]LY335979 showed that Pgp had aK _d of 73 nM, which is in good agreement with the previously determined K _i value. Thus, LY335979 is an extremely potent Pgp, and not MRP1 or MRP2, modulator and has a significantly lower affinity for CYP3A than for Pgp.