17beta-Hydroxysteroid dehydrogenases (17beta-HSD) regulate the intracellular concentration of active sex steroid hormones in target tissues. To date, at least 14 different isozymes have been identified. The type 8 17beta-hydroxysteroid dehydrogenase (17beta-HSD8) selectively catalyzes the conversion of estradiol (E2) to estrone (E1). To map the promoter region and to investigate its regulation, we cloned and fused a 1600 bp DNA fragment upstream of the 17beta-HSD8 transcriptional start site to a luciferase reporter gene. After transient transfection in HepG2 cells, this fragment was shown to possess promoter activity. Deletion constructs of the 5' flanking region of the 17beta-HSD8 gene led to the identification of the minimal promoter region within the first 75 bp upstream of the transcriptional start site. This region included two CCAAT boxes and sequences closely resembling the consensus Sp1 and NF-kappaB motifs. Site directed mutagenesis revealed that the CCAAT boxes were essential for transcription in HepG2. EMSA, supershift and chromatin immunoprecipitation reflected that these sequences were binding sites for C/EBPbeta. Furthermore, promoter activity was increased by the co-transfection of a C/EBPbeta expression vector, and this transactivation was through both CCAAT boxes. Our studies indicate that C/EBPbeta is essential for the transcription of the 17beta-HSD8 gene in the liver.