Despite the strong conservation of proximal 5'-flanking DNA sequences, cell transfection and transgenic animal studies have failed to provide a unifying hypothesis to explain the expression of both mouse and human renin genes. Recently, sequences contained in the mouse Ren-1c gene 5'-flanking DNA (-2866 to -2625) were shown to contain an enhancer-like element that stimulates Ren-1c promoter activity in renin-expressing As4.1 cells approximately 80-fold. Earlier studies using transgenic mice had suggested that this same region is required for the cell-specific expression of mouse renin genes. Since existing human renin genomic clones lack sequences homologous to the mouse renin enhancer, we isolated several human P1 and P1 artificial chromosome genomic clones that contain > 80 kb spanning the human renin gene. Analysis of these clones by Southern blot hybridization and long-rang polymerase chain reaction showed that they contain sequences homologous to the mouse enhancer at approximately 12 kb upstream of the transcription start site. Mouse and human sequences were 59% identical over a 650-bp region that contained the minimal enhancer from the mouse Ren-1c gene. However, a 1-kb fragment containing the entire human enhancer homology failed to stimulate human renin promoter activity in transiently transfected As4.1 cells. Further deletional analysis showed that a 220-bp region of the human sequence highly conserved in the mouse Ren-1c gene exhibited up to 47-fold transcriptional stimulation, although this was lower than the maximal effect exhibited by the minimal mouse enhancer (223-fold). Taken together, these observations suggest that sequences surrounding the conserved enhancer core stimulate enhancer activity in the mouse gene but suppress activity in the human gene. The high transcriptional activity of the mouse enhancer may have evolved to support the exceptionally high plasma renin concentrations found in mice. However, the enhancer core and surrounding conserved sequences may play an additional role in directing cell specificity.