Increases in gene dosage of the transcriptional regulatory protein yAP-1 in Saccharomyces cerevisiae, causes profound hyperresistance to a myriad of cytotoxic agents. 3-Amino-1,2,4-triazole (3-AT) and 4-nitroquinoline-N-oxide (4-NQO) are two such cytotoxic compounds. The ATR1 locus is a putative membrane bound protein that belongs to the major facilitator superfamily (MFS), is regulated by Gcn4 and was cloned by its ability to confer resistance to 3-AT and 4-NQO. We show here that the ATR1 gene is necessary for yAP-1-mediated resistance to 3-AT and 4-NQO. ATR1 expression is responsive to YAP1 gene dosage and the ATR1 promoter region contains a yAP-1 response element (YRE) 250 bp upstream of transcription start. The ATR1 YRE (YREA) contains 11/14 identities with the previously identified dYCF1 YRE. DNase I footprinting using a probe containing the YREA showed that bacterially-produced yAP-1 and Gcn4, were able to bind this element. Furthermore, a site-directed mutation in the YREA that abolished yAP-1 and Gcn4, binding din vitro prevented the mutant promoter from responding to differing YAP1 gene dosage. A mutant ATR1 gene constructed with this point mutation in the YREA was hypersensitive to 3-AT and 4-NQO. These data implicate the bZip transcription factors Gcn4 and yAP-1 in the co-regulation of ATR1.