The renin gene is regulated by an enhancer located 2.6 kb upstream of the transcription start site in the mouse and 11 kb upstream in humans. Despite extensive sequence conservation, the mouse renin enhancer is transcriptionally more active than the human renin enhancer. We report that the mechanism accounting for this is a result of sequence variation in the promoter proximal half-site of a retinoic-acid response element present in the enhancer. This sequence difference also prompted us to search for naturally occurring polymorphisms in the renin enhancer among normal and hypertensive human subjects. We sequenced the kidney enhancer from 90 samples derived from the Coriell Polymorphism Discovery Resource and 95 severely hypertensive Caucasian and African-American individuals. A single relatively frequent polymorphism (7, 2, and 7%, respectively in the Coriell, African-American, and Caucasian) was identified in the enhancer, one nucleotide downstream of the promoter distal half-site of the retinoic-acid response element. This variant was transcriptionally silent in transfection assays performed in renin-expressing As4.1 cells, a model of renal juxtaglomerular cells. A singleton polymorphism in the promoter was also identified in a single African-American individual. This polymorphism was located between binding sites for CBF1 and homeobox D10 but was also transcriptionally silent either in the presence or absence of the enhancer. Our study demonstrates the presence of silent polymorphisms in the renin promoter and enhancer, thus underscoring the critical importance of performing functional analyses before initiating expensive clinical studies seeking association between polymorphisms and complex diseases such as hypertension.