A human MDR1 cDNA was introduced into yeast cells. Immunoblot analysis and indirect immunostaining showed that some of the P-glycoprotein produced was situated in its native orientation in the yeast plasma membrane. Drug-binding activities of the recombinant P-glycoproteins were markedly decreased compared to that of the authentic P-glycoprotein. To identify the bases of decreased binding we studied the effects of membrane component sterols on the azidopine binding and found that ergosterol, which is the main sterol in the yeast membrane, and calciferol, which is produced from ergosterol by UV irradiation, inhibited azidopine binding. These sterols in yeast membrane probably inhibit the function of human P-glycoprotein as a multidrug transporter in yeast cells, because expression of P-glycoprotein in yeast cells did not confer resistance to doxorubicin.