CYP2A8 is a major form of cytochrome P-450 inducible by 3-methylcholanthrene in Syrian hamster liver. To identify DNA elements necessary for the transcriptional activation of the CYP2A8 gene, we analyzed the regulatory region of the CYP2A8 gene and conducted transient transfection experiments of CYP2A8-luciferase fusion plasmids in primary cultures of hamster hepatocytes. We analyzed up to -5 kb of the 5'-flanking region and found the region sufficient for the 3-methylcholanthrene-inducible gene expression. This region contained a consensus sequence for xenobiotic responsive element (XRE) between -2366 and -2349, which was shown to be essential for induction of the gene expression. Furthermore, we found a novel positive regulatory element for XRE-mediated gene expression (PREX) located upstream of the XRE. This element is not identified in any genes inducible by 3-methylcholanthrene so far reported. Without PREX, the XRE-mediated promoter activity was enhanced nearly 10-fold, whereas with PREX, the activity was enhanced 20-fold over the basal level. Gel mobility shift assays revealed specific binding of nuclear proteins to PREX. Mutations and deletions of PREX caused a loss of the binding and promoter-enhancing activities, respectively. Moreover, transient expression experiments showed that the enhancing activity of PREX was not observed in Drosophila Schneider's line 2 cells, which were shown to lack the PREX binding proteins.