Multidrug resistance in yeast results from over-expression of drug efflux transporter genes due to gain-of-function mutations in transcription factors. To suppress multidrug resistance at the level of gene expression, we have developed a yeast-based screening system for the detection of compounds down-regulating the major multidrug ABC transporter Pdr5p expressed under the control of Pdr3p transcription factor. Here, we report the construction and properties of the improved set of yeast strains designed along with such screening also for a global analysis of genetic suppressors of multidrug resistance. The basic components of this system, the P GAL1-PDR3 and P PDR5-pma1(D378N) fusion genes, were individually or simultaneously integrated into corresponding chromosomes of a hypersensitive S. cerevisiae strain deleted in the PDR1 and PDR3 genes. This resulted in increased mitotic stability of a set of new test strains compared with the original prototrophic strain ZK11-1 developed previously. In addition, some of the strains designed are auxotrophic for leucine, uracil and histidine allowing them to be used in genetic screens for positive selection of multicopy or loss-of-function genetic suppressors of multidrug resistance.