Essential hypertension is a common disease the genetic determinants of which have been difficult to unravel because of its clinical heterogeneity and complex, multifactorial, polygenic etiology. Based on our observations that α1-Na,K-ATPase (ATP1A1) and renal-specific, bumetanide-sensitive Na, K,2Cl-cotransporter (NKCC2) genes interactively increase susceptibility to hypertension in the Dahl salt-sensitive hypertensive (Dahl S) rat model, we investigated whether parallel molecular genetic mechanisms might exist in human essential hypertension in a relatively genetic homogeneous cohort in northern Sardinia. Putative ATP1A1-NKCC2 gene interaction was tested by comparing hypertensive patients (blood pressure [BP] >165/95 mm Hg) with normotensive controls age >60 years with BP <140/85 mm Hg. Genotype analysis with microsatellite markers revealed conformation to Hardy-Weinberg proportions for 6 alleles of both ATP1A1 (D1S453) and NKCC2 (NKCGT7) markers, respectively. Two-by-six X2 analysis of alleles identified overrepresentation of ATP1A1 No. 4 and NKCC2 No. 4 alleles, respectively, in hypertensives compared with controls. With a qualitative trait framework, single-gene analysis detected association of both the ATP1A1 No. 4 allele (P=0.004, X2=8.094, df=1) and the NKCC2 No. 4 allele (P=0.0002, X2=14.279, df=1) with moderate to severe hypertension. Digenic analysis revealed that ATP1A1 No. 4-NKCC2 No. 4 allele interaction increases susceptibility to hypertension (P<0.0001, X2=22.3, df=1) beyond levels obtained in single-gene analysis. Analysis was also performed in a quantitative trait framework with BP as the continuous trait parameter. Digenic analysis of ATP1A1 No. 4-NKCC2 No. 4 allele interaction revealed significant association with systolic (1-way ANOVA, P=0.000076) and diastolic (P=0.00099) BP. Interaction was corroborated by 2×2 factorial ANOVA for interaction (systolic BP interaction term, P<0.05, diastolic BP interaction term, P=0.035). The data are compelling that ATP1A1 and NKCC2 genes are candidate interacting hypertension-susceptibility loci in human essential hypertension and affirm gene interaction as an important genetic mechanism underlying hypertension susceptibility. Although corroboration in other cohorts and identification of functionally significant mutations are imperative next steps, the data provide a genotype-stratification scheme, with 4-fold predictive value (odds ratio, 4.28; 95% confidence interval, 2.29 to 8.0), which could help decipher the complex genetics of essential hypertension.