As our understanding of the contributory roles of NO in the blood vessel wall evolves, so does the need to firmly understand the basic principles governing the regulated expression of the endothelial nitric oxide synthase (eNOS) gene. Because a robust approach to dissecting the relative contribution of a given cardiovascular gene exploits the use of murine genetic models, P1 murine genomic clones were isolated, characterized and functionally assessed to gain further insight into the regulated expression of the eNOS gene in the mouse. Sequence analysis of 1.8 kb of 5' flanking regions revealed important regions of sequence conservation with human and bovine sequences. Functional promoter activity was confirmed using transient transfection analysis of cultured endothelial cells.