A gene from Saccharomyces cerevisiae whose overexpression confers resistance to 10-N-nonyl acridine orange (NAO) has been isolated. This cationic dye binds acidic phospholipids and more specifically cardiolipin (Petit, J. M., Maftah, A., Ratinaud, M. H. and Julien, R. Eur. J. Biochem. 209, 267-273, 1992). The isolated gene was found to be identical to SGE1, a partial multicopy suppressor of the gal11 mutation (Amakasu, H., Suzuki, Y., Nishizawa, M. and Fukasawa, T. Genetics 134, 675-683, 1993), that also confers crystal violet resistance to a supersensitive strain (Ehrenhofer-Murray, A. E., Wurgler, F. E. and Sengstag, C. Mol. Gen. Genet. 244, 287-294, 1994). The data presented in this paper show that the SGE1 gene product, a member of the major facilitator superfamily, confers a pleiotropic drug-resistance phenotype when present in high copy number. The results also demonstrate that Sge1p acts as an extrusion permease whose specificity seems restricted to dye molecules possessing a large unsaturated domain that stabilizes a permanent positive charge such as NAO, crystal violet, ethidium bromide or malachite green.