We analyzed the phenotype associated with the atrial natriuretic peptide (ANP) genetic variant rs5065 in a random community-based sample. We also assessed and compared the biological action of 2 concentrations (10 −10 mol/L, 10 –8 mol/L) of ANP and ANP-RR, the protein variant encoded by the minor allele of rs5065, on activation of the guanylyl cyclase (GC)-A and GC-B receptors, production of the second messenger 3′,5′-cGMP in endothelial cells, and endothelial permeability. rs5065 genotypes were determined in a cross-sectional adult cohort from Olmsted County, MN (n=1623). Genotype frequencies for rs5065 were 75%, 24%, and 1% for TT, TC, and CC, respectively. Multivariate analysis showed that the C allele was associated with increased risk of cerebrovascular accident (hazard ratio, 1.43; 95% confidence interval, 1.09–1.86; P =0.009) and higher prevalence of myocardial infarction (odds ratio, 1.82; 95% confidence interval, 1.07–3.09; P =0.026). ANP-RR 10 −8 mol/L activated the GC-A receptor (83.07±8.31 versus no treatment 0.18±0.04 per 6 wells; P =0.006), whereas ANP-RR 10 −10 mol/L did not. Neither 10 −8 mol/L nor 10 −10 mol/L ANP-RR activated GC-B receptor ( P =0.10, P =0.35). ANP 10 −8 mol/L and ANP-RR 10 −8 mol/L stimulated 3′,5′-cGMP production in endothelial cells similarly ( P =0.58). Both concentrations of ANP-RR significantly enhanced human aortic endothelial cell permeability (69 versus 29 relative fluorescence units [RFUs], P =0.012; 58 versus 39 RFUs, P =0.015) compared with ANP. The minor allele of rs5065 was associated with increased cardiovascular risk. ANP-RR activated the GC-A receptor, increased 3′,5′-cGMP in endothelial cells, and when compared with ANP, augmented endothelial cell permeability.