Multiple or pleiotropic drug resistance in the yeast Saccharomyces cerevisiae requires the expression of several ATP binding cassette transporter-encoding genes under the control of the zinc finger-containing transcription factor Pdr1p. The ATP binding cassette transporter-encoding genes regulated by Pdr1p include PDR5 and YOR1, which are required for normal cycloheximide and oligomycin tolerances, respectively. We have isolated a new member of the PDR gene family that encodes a member of the Hsp70 family of proteins found in this organism. This gene has been designated PDR13 and is required for normal growth. Overexpression of Pdr13p leads to an increase in both the expression of PDR5 and YOR1 and a corresponding enhancement in drug resistance. Pdr13p requires the presence of both the PDR1 structural gene and the Pdr1p binding sites in target promoters to mediate its effect on drug resistance and gene expression. A dominant, gain-of-function mutant allele of PDR13 was isolated and shown to have the same phenotypic effects as when the gene is present on a 2μm plasmid. Genetic and Western blotting experiments indicated that Pdr13p exerts its effect on Pdr1p at a posttranslational step. These data support the view that Pdr13p influences pleiotropic drug resistance by enhancing the function of the transcriptional regulatory protein Pdr1p.