Multiple or pleiotropic drug resistance in the yeast Saccharomyces cerevisiae can arise from overexpression of the Pdr5 and Snq2 ATP binding cassette multidrug transporters. Expression of Pdr5 and Snq2 is regulated by the two transcription factors Pdr1 and Pdr3, as multidrug-resistant pdr1 and pdr3 gain-of-function mutants overexpress both drug efflux pumps. One such pdr1 mutant allele was previously cloned in a genetic screen by its ability to suppress the squelching toxicity mediated by an estradiol-inducible chimeric VP16-human estrogen receptor (VEO) expressed in yeast (Gilbert, D. M. , Heery, D. M., Losson, R., Chambon, P., and Lemoine, Y. (1993) Mol. Cell. Biol. 13, 462-472). In this study, we demonstrate that relief of estradiol toxicity in yeast cells expressing VEO requires functional PDR5 and SNQ2 genes, since a Deltapdr5 Deltasnq2 double deletion leads to an increased estradiol toxicity. Furthermore, using URA3 as an estradiol-inducible reporter gene, we show that Pdr5 and Snq2, when overexpressed from high-copy plasmids, can reduce the intracellular concentration of estradiol. In contrast, a Deltapdr5 Deltasnq2 double deletion mutant accumulates almost 30-fold more intracellular estradiol than the isogenic wild type. Indirect immunofluorescence showed that a pdr1-3 mutant massively overexpresses Pdr5 at the plasma membrane, suggesting that estradiol efflux from the cells occurs across the plasma membrane. Our data demonstrate that Pdr5 and Snq2 can transport steroid substrates in vivo and suggest that steroids and/or related membrane lipids could represent physiological substrates for certain yeast ABC transporters, which are otherwise involved in the development of pleiotropic drug resistance.