Growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid results in the induction of the ATP-binding cassette (ABC) transporter Pdr12 in the plasma membrane (P. Piper, Y. Mahe, S. Thompson, R. Pandjaitan, C. Holyoak, R. Egner, M. Muhlbauer, P. Coote, and K. Kuchler, EMBO J. 17:4257-4265, 1998). Pdr12 appears to mediate resistance to water-soluble, monocarboxylic acids with chain lengths of from C(1) to C(7). Exposure to acids with aliphatic chain lengths greater than C(7) resulted in no observable sensitivity of Deltapdr12 mutant cells compared to the parent. Parent and Deltapdr12 mutant cells were grown in the presence of sorbic acid and subsequently loaded with fluorescein. Upon addition of an energy source in the form of glucose, parent cells immediately effluxed fluorescein from the cytosol into the surrounding medium. In contrast, under the same conditions, cells of the Deltapdr12 mutant were unable to efflux any of the dye. When both parent and Deltapdr12 mutant cells were grown without sorbic acid and subsequently loaded with fluorescein, upon the addition of glucose no efflux of fluorescein was detected from either strain. Thus, we have shown that Pdr12 catalyzes the energy-dependent extrusion of fluorescein from the cytosol. Lineweaver-Burk analysis revealed that sorbic and benzoic acids competitively inhibited ATP-dependent fluorescein efflux. Thus, these data provide strong evidence that sorbate and benzoate anions compete with fluorescein for a putative monocarboxylate binding site on the Pdr12 transporter.