Multiple or pleiotropic drug resistance most often occurs in Saccharomyces cerevisiae due to substitution mutations within the Cys6-Zn(II) transcription factors Pdr1p and Pdr3p. These dominant transcriptional regulatory proteins cause elevated drug resistance and overexpression of the ATP-binding cassette transporterencoding gene, PDR5. We have carried out a genetic screen to identify negative regulators of PDR5 expression and found that loss of the mitochondrial genome (ρ°cells) causes up-regulation of Pdr3p but not Pdr1p function. Additionally, loss of the mitochondrial inner membrane protein Oxa1p generates a signal that results in increased Pdr3p activity. Both of these mitochondrial defects lead to increased expression of the PDR3 structural gene. Importantly, the signaling pathway used to enhance Pdr3p function in ρ°cells is not the same as in oxal cells. Loss of previously described nuclear-mitochondrial signaling genes like RTG1 reduce the level of PDR5 expression and drug resistance seen in ρ°cells but has no effect on oxa1-induced phenotypes. These data uncover a new regulatory pathway connecting expression of multidrug resistance genes with mitochondrial function.