P-glycoprotein, the multidrug transporter, is proposed to act as an ATP-driven drug efflux pump. We previously reported the partial purification of P-glycoprotein from multidrug-resistant cells (Doige, C. A., Yu, X., and Sharom, F. J. (1992) Biochim. Biophys. Acta 1109, 149-160). We now report the reconstitution of this preparation into phospholipid bilayers using rapid detergent removal by gel filtration chromatography. The resulting proteoliposomes displayed ATP-dependent [3H]colchicine uptake over a time period of 0-4 min. No drug uptake was observed for liposomes of lipid alone, or liposomes reconstituted with a similar extract from drug-sensitive cells. Drug uptake was osmotically sensitive, and abolished by detergent permeabilization, indicating that it represented true transport into the vesicle lumen. Steady-state levels of drug uptake increased with drug concentration, approaching saturation at approximately 150 microM colchicine, with half-maximal accumulation at 50 microM. Drug was accumulated actively against a 5.6-fold concentration gradient. Multidrug resistance spectrum drugs and chemosensitizers inhibited colchicine uptake by P-glycoprotein proteoliposomes, whereas cytosine arabinoside and methotrexate had no effect. Reconstituted liposomes showed high levels of ATPase activity, which was stimulated over 2-fold by verapamil and trifluoperazine. These results suggest that P-glycoprotein functions as an active drug transporter with constitutive ATPase activity.