High-level expression of P-glycoprotein, a 170-kDa mammalian plasma membrane ATPase, is the cause of an important and widespread form of cancer multidrug resistance. P-glycoprotein reduces cellular accumulation of an enormous variety of lipophilic compounds. The basis for this broad substrate specificity is not well understood. We explored this issue by measuring the kinetics of transport of the lipophilic P-glycoprotein substrate Hoechst 33342 by P-glycoprotein-rich plasma membrane vesicles from CH(R)B30 cells. Hoechst 33342 is fluorescent when bound to the membrane, but not when in the aqueous medium, allowing movement of the dye out of the membrane to be quantitated by fluorescence intensity. The initial specific rate of transport was directly proportional to the amount of Hoechst 33342 in the lipid phase and inversely proportional to the concentration in the aqueous phase. This demonstrates that P-glycoprotein removes Hoechst 33342 from the lipid membrane, where it concentrates due to its hydrophobicity. Because the membrane concentration of hydrophobic P-glycoprotein substrates is high, it may be that P-glycoprotein need not recognize them with high affinity. Transport of hydrophobic substrates out of the lipid bilayer instead of the cytoplasm thus helps to explain the broad substrate specificity of P-glycoprotein.