gamma-Glutamyl transpeptidase is normally not present in adult rat hepatocytes, but its expression is induced by a range of xenobiotics, including carcinogens and chemopreventive agents. Synthesis of the enzyme is mediated by at least six mRNAs transcribed from tandemly arranged promoters on a single gene. We previously identified and partially characterized promoter III as being responsible for upregulation of gamma-glutamyl transpeptidase in rat liver in response to inducing agents. In this study, we examined response elements involved in the regulation of this promoter by using reporter gene assays and in vitro DNase I footprinting and electrophoretic mobility shift assays. Among the response elements was a region with negative regulatory activity upstream of a 240-bp basal regulatory region covering the transcriptional start site. This negative regulatory region, lying between nt -465 and -185, contained sequences with considerable homology to silencer elements in the glutathione s-transferase P gene. The region of basal regulation (nt -185 to +55) contained a CCAAT box, a TFIID binding site, and a GAGA box. A hepatocyte nuclear factor 3-like sequence was identified at nt -234 to -254 and had a DNase I hypersensitive site characteristic of binding of members of the hepatocyte nuclear factor 3/fork head family of proteins and may be involved in the regulation of this promoter.