The catalytic reaction of renin, an aspartyl proteinase, with angiotensinogen is the rate-limiting step fo the renin-angiotensin system involved in the maintenance of blood pressure and electrolyte balance in mammals. We have characterized species-specific expression of the hepatic renin gene by RNase protection experiment, primer extension analysis, and promoter assay using an in vitro DNA transfection. RNase protection experiments revealed that the renin gene is expressed in rat liver, but neither in mouse nor in human. Primer extension analysis identified the putative promoter region of the rat renin gene, which contains TATAAAA sequence, a canonical regulatory DNA element. In order to test whether the upstream region of the renin gene with respect to the putative transcription initiation site is a functional promoter, we have examined the ability of the 5'-flanking sequences of the rat renin gene as well as the human and mouse genes to activate expression of a reporter gene containing the bacterial chloramphenicol acetyltransferase (CAT)-coding sequences, by transient transfection assays. In transfected HepG2 cells, a hepatoma cell line, only the rat renin promoter was capable of driving the CAT gene expression. These results suggested that the rat-specific renin gene expression in the liver could be primarily determined by its promoter specificity.