The expression of a mu-class glutathione S-transferase gene (hGSTYBX) isolated from hamster smooth muscle tumor cells (DDT1 MF-2) is transcriptionally up-regulated by glucocorticoids, and this hormonal regulation is dependent upon protein synthesis. To study the mechanism of regulation, we have cloned and sequenced hGSTYBX genomic DNA including its 5' flanking region. The hGSTYBX gene contains nine exons dispersed over a 6.3-kilobase region. When linked to a chloramphenicol acetyltransferase (CAT) reporter gene, the 5' flanking region was able to direct transcription of the reporter gene. With 5' deletion studies, we have localized the major glucocorticoid-inducible regulatory element between nucleotides -353 and -239. Within this region no classic glucocorticoid response element (TGTTCT) was identified, but four potential helix-loop-helix binding domains are embedded in two 16-base-pair repeats. Another glucocorticoid regulatory domain has been localized between nucleotides -239 and -136. Cycloheximide blocks glucocorticoid-induced transcription of both the -353CAT and -239CAT reporter genes (nucleotides -447 to -12 and nucleotides -239 to -12 of hGSTYBX, respectively, ligated to a CAT reporter gene); therefore, our observations support previous results suggesting that hGSTYBX induction by glucocorticoids is a secondary response.