Multidrug-resistant (MDR) tumor cells reduce the toxicity of antineoplastic drugs by an energy-dependent active efflux mechanism mediated by the MDR1 gene product, the P-glycoprotein (Pgp). Pgp expressed in cultured Sf9 insect cells has been shown to exhibit a high capacity ATPase activity in the presence of a variety of drugs known to be transported by the Pgp (Sarkadi et al., J Biol Chem 267: 4854-4858, 1992). The strict dependence of the Pgp ATPase activity on the presence of transport substrates indicates that the drug-stimulated ATPase activity is a direct reflection of the drug transport function of the Pgp. In the present study, this system has been utilized to investigate the possibility that antiestrogens and steroid hormones are transported by the Pgp. Antiestrogens such as tamoxifen, metabolites of tamoxifen (4-hydroxytamoxifen and N-desmethyltamoxifen), droloxifen, and toremifene stimulated the Pgp ATPase activity, and the maximum stimulation obtained with these agents equalled the maximal stimulation obtained by the best known MDR chemosensitizer, verapamil. Clomifene, nafoxidine and diethylstilbestrol also stimulated the Pgp ATPase activity, with maximal activations 75, 60 and 45% of the verapamil stimulation, respectively. Different degrees of stimulation of the Pgp ATPase activity were also obtained in the presence of steroid hormones such as progesterone, beta-estradiol, hydrocortisone, and corticosterone. Among these, progesterone is a potent inducer of the Pgp ATPase activity; at 50 microM, this hormone stimulated the Pgp ATPase activity as effectively as verapamil. These results suggest that the antiestrogens and steroid hormones that are known to reverse the multidrug-resistant phenotype do so by directly interacting with Pgp, thus interfering with its anticancer drug-extruding activity.