Through the genotype/phenotype cosegregation analysis of an F2intercross, from the crossbreeding of stroke-prone spontaneously hypertensive rats (SHRSP) and stroke-resistant spontaneously hypertensive rats (SHR), we previously identified a quantitative trait locus for stroke on rat chromosome 5 (STR2) that colocalized with the genes encoding atrial and brain natriuretic peptides (ANPandBNP) and conferred a stroke-delaying effect. To further characterizeANPandBNPas candidates for stroke, we performed additional studies. Comparative sequence analysis revealed point mutations in both the coding and regulatory regions ofANP, whereas no interstrain differences were found forBNP. In in vitro studies in COS-7 and AtT-20 cells that were performed to test the relevance of a G→A substitution at position 1125, a Gly→Ser transposition in the SHRSP pro-ANP peptide resulted in different posttranslational processing of the SHRSP ANP gene product that was also associated with higher cGMP production (P<0.05). Furthermore, an analysis of a 5′ end mutation affecting a PEA2 regulatory binding site in the 5′ untranslated regulatory sequence of SHRSPANPdemonstrated a significantly lowerANPpromoter activation in endothelial cells (P<0.05 versus the SHRANP). In addition, the expression ofANPwas significantly reduced in the brain, but not in the atria, of SHRSP compared with SHR (P<0.0001). No differences were detected with regard toBNPexpression. The present results reveal substantial differences inANP, but notBNP, structure and product among SHR and SHRSP, which supports a role ofANPin the pathogenesis of stroke in the SHRSP animal model.