The Cyp4b1 structural gene comprises 12 exons that span approximately 23kb. The transcriptional initiation site, mapped by 5(')-RACE and primer extension analyses from kidney mRNA, was found to be 34-bp upstream of the translation initiation site. Cyp4b1 mRNA expression was found restricted to kidney and androgenic up-regulation in several mouse strains was observed. Spatial location investigated by in situ hybridisation revealed abundant and specific expression of Cyp4b1 mRNA in the proximal tubules of the renal cortex. A 1.8-kb murine Cyp4b1 5(')-flanking region, which encompasses a TATA box-like sequence and several putative transcription factor-binding sites, was isolated. Transient transfection studies with different Cyp4b1 promoter constructs indicated that this flanking region exhibits promoter activity when expressed in different cell lines and that a 98-truncated fragment represents the minimal sequence required for basal transcription. Androgen responsiveness was examined by cotransfection with an androgen receptor expression vector, in the presence of androgens.