The antineoplastic alkaloid camptothecin interferes with the catalytic cycle of DNA topoisomerase I rendering it a cellular poison. Camptothecin stabilizes a covalent enzyme-DNA intermediate that is converted into lethal double strand DNA lesions during S phase of the cell cycle. Yeast SCT1 mutants were isolated in a screen for mutations in genes other than TOP1 that result in camptothecin resistance. Here we report SCT1 is allelic to PDR1 and that a Thr-879 to Met substitution in the PDR1-101 transcription factor confers multiple drug resistance. PDR1 regulates the expression of several gene products including the ATP-binding cassette transmembrane transport proteins PDR5, YOR1, and SNQ2. The PDR1 T879M mutant increased PDR5 transcription compared with wild-type PDR1 strains. Deletion of PDR1 or the downstream effector SNQ2 increased cell sensitivity to camptothecin, whereas deletion of YOR1 or PDR5 had little effect on camptothecin sensitivity. However, the camptothecin resistance accompanying GAL1-promoted overexpression of PDR5 suggests some substrate promiscuity among the ATP- binding cassette transporters. These data underscore the role of the pleiotropic drug resistance network in regulating camptothecin toxicity and are consistent with a model of decreased intracellular concentrations of camptothecin resulting from the increased expression of the SNQ2 transporter.