We recently found that recombinant NBD1 cytosolic domain corresponding to segment 395-581 of mouse mdr1 P-glycoprotein bound fluorescent 2'(3')-N-methylanthraniloyl-ATP (MANT-ATP) with high affinity [Dayan, G., Baubichon-Cortay, H., Jault, J.-M., Cortay, J. -C., Deléage, G., & Di Pietro, A. (1996) J. Biol. Chem. 271, 11652-11658]. The present work shows that a longer 371-705 domain (extended-NBD1), including tryptophan-696 as an intrinsic probe, which bound MANT-ATP with identical affinity, also interacted with steroids known to modulate anticancer drug efflux from P-glycoprotein-positive multidrug-resistant cells. Progesterone, which is not transported, its hydrophobic derivatives medroxyprogesterone acetate and megestrol acetate, and Delta6-progesterone produced nearly a 50% saturating quenching of the domain intrinsic fluorescence, with dissociation constants ranging from 53 to 18 microM. The even more hydrophobic antiprogestin RU 486 produced a complete quenching of tryptophan-696 fluorescence, in contrast to more hydrophilic derivatives of progesterone containing hydroxyl groups at positions 11, 16, 17, and 21 and known to be transported, which produced very little quenching. A similar differential interaction was observed with full-length purified P-glycoprotein. The steroid-binding region within extended-NBD1 appeared distinct from the nucleotide-binding site as the RU 486-induced quenching was neither prevented nor reversed by high ATP concentrations. In contrast, MANT-ATP binding was efficiently prevented or displaced by RU 486, suggesting that the hydrophobic MANT group of the bound nucleotide analogue overlaps, at least partially, the adjacent steroid-binding region revealed by RU 486.