A distal transcriptional enhancer has been previously reported upstream of the mouse renin gene. A homologous sequence is also present upstream of the human renin gene, but the mouse and human renin enhancers differ markedly in their ability to activate transcription of a renin promoter. Although the 2 enhancers share high homology in their 202-bp promoter distal portions, their 40-bp proximal portions are heterogeneous. Chimeric enhancers were used to test the role of the 40-bp segment (m40) of the enhancer by using transient transfection analysis in mouse kidney renin-expressing As4. 1 cells. Deletion of m40 from the mouse renin enhancer or its addition to the human renin enhancer did not significantly change transcriptional activity when placed close to a mouse or human renin promoter. However, when placed further upstream of a renin promoter, the same deletion and substitution markedly altered enhancer activity. Electrophoretic gel mobility shift analysis identified 2 elements, a and b, in m40 that specifically bound nuclear proteins from As4.1 cells. Mutagenesis and transient transfection analysis revealed that element b accounts for the function of m40 and that element a antagonizes the positive influence of element b. Gel competition and supershift analysis revealed that nuclear factor-Y, a ubiquitous CAAT-box binding protein, binds to element a. Sequence analysis revealed that the human renin enhancer has a natural loss-of-function mutation in element b that affects its ability to transactivate when placed far upstream of a promoter. Reversion of the human renin element b to match the mouse sequence restored transactivation of the enhancer in mouse As4.1 cells. These data suggest that element b cooperates with the rest of the enhancer to maintain full enhancer activity, whereas element a may regulate enhancer activity. Sequence differences in these elements may explain the functional differences in the mouse and human renin enhancer sequences.