Rhodamine 123 is a mitochondrial dye that is retained for prolonged periods by carcinoma cells. While investigating causes of retention of this dye, we found that 10 microM progesterone caused a rapid stimulation of efflux of rhodamine 123 within 15 min from KB V20C cells, which overexpress the multidrug resistance pump. Progesterone did not stimulate efflux from KB cells that do not overexpress the pump, and verapamil blocked rhodamine 123 efflux in the presence or absence of progesterone, indicating that rhodamine 123 is removed from KB V20C cells by the multidrug resistance pump. Progesterone, however, is unlikely to stimulate rhodamine 123 efflux by simply increasing pump activity for two reasons: (1) progesterone inhibited the efflux of daunomycin from KB V20C cells, so it did not stimulate efflux of all drugs, and (2) progesterone inhibited efflux of rhodamine 123 from L1210/VMDRC cells and had little effect on Adr MCF7 cells; both overexpress the multidrug resistance pump. In the experiments with KB V20C cells, progesterone was the most active steroid tested. At 10 microM, progesterone caused a 70-fold stimulation, desoxycorticosterone, testosterone, promegestone and estradiol about 20-fold, and others had little or no effect. Progesterone may act by a non-genomic mechanism to decrease intracellular binding of rhodamine 123, making the dye accessible to the multidrug resistance pump.