Glutathione-dependent detoxification reactions are catalyzed by the enzyme glutathione S-transferase and are important in drug resistance in organisms ranging from bacteria to humans. The yeast Issatchenkia orientalis expresses a glutathione S-transferase (GST) protein that is induced when the GST substrate o-dinitrobenzene (o-DNB) is added to the culture. In this study, we show that overproduction of the I. orientalis GST in Saccharomyces cerevisiae leads to an increase in o-dinitrobenzene resistance in S. cerevisiae cells. To recover genes that influence o-DNB resistance in S. cerevisiae, a high copy plasmid library was screened for loci that elevate o-DNB tolerance. One gene was recovered and designated ROD1 (resistance to o-dinitrobenzene). This locus was found to encode a novel protein with no significant sequence similarity with proteins of known function in the data base. An epitope-tagged version of Rod1p was produced in S. cerevisiae and shown to function properly. Subcellular fractionation experiments indicated that this factor was found in the particulate fraction by differential centrifugation. Overproduction of Rod1p leads to resistance to not only o-DNB but also zinc and calcium. Strains that lack the ROD1 gene are hypersensitive to these same compounds. Rod1p represents a new type of molecule influencing drug tolerance in eukaryotes.