P-Glycoprotein (P-GP) plays a pivotal role in maintaining the multidrug-resistant (MDR) phenotype. This membrane glycoprotein is overproduced in MDR cells and the endometrium of the mouse gravid uterus (Arceci, R. J., Croop, J. M., Horwitz, S. B., and Housman, D. (1988) Proc. Natl. Acad. Sei. U. S. A. 85, 4350–4354). This latter observation and an interest in endogenous substrates for P-GP led to a study of the interaction of steroids with P-GP found in the endometrium of the mouse gravid uterus and in MDR cells derived from the murine macrophage-like cell J774.2. [³H]Azidopine labeling of P-GP from these two sources was inhibited by various steroids, particularly progesterone. Progesterone also markedly inhibited [³H]vinblastine binding to membrane vesicles prepared from MDR cells, enhanced vinblastine accumulation in MDR cells, and increased the sensitivity of MDR cells to vinblastine. In addition, we have demonstrated that the hydrophobicity of a steroid is important in determining its effect on inhibition of drug binding to P-GP. It is concluded that progesterone, a relatively nontoxic endogenous steroid, interacts with P-GP and is capable of reversing drug resistance in MDR cells.