Improper control of expression of ATP binding cassette transporter- encoding genes is an important contributor to acquisition of multidrug resistance in human tumor cells. In this study, we have analyzed the function of the promotor region of the Saccharomyces cerevisiae YOR1 gene, which encodes an ATP binding cassette transporter protein that is required for multidrug tolerance in S. cerevisiae. Deletion analysis of a YOR1-lacZ fusion gene defines three important transcriptional regulatory elements. Two of these elements serve to positively regulate expression of YOR1, and the third element is a negative regulatory site. One positive element corresponds to a Pdr1p/Pdr3p response element, a site required for transcriptional control by the homologous zinc finger transcription factors Pdr1p and Pdr3p in other promoters. The second positive element is located between nucleotides -535 and -299 and is referred to as UAS(YOR1) (where UAS is upstream activation sequence). Interestingly, function of UAS(YOR1) is inhibited by the downstream negative regulatory site. Promoter fusions constructed between UAS(YOR1) and the PDR5 promoter, another gene under Pdr1p/Pdr3p control, are active, whereas analogous promoter fusions constructed with the CYC1 promoter are not. This suggests the possibility that UAS(YOR1) has promoter-specific sequence requirements that are satisfied by another Pdr1p/Pdr3p-regulated gene but not by a heterologous promoter.