Interactions or epistasis between genetic factors may contribute to "missing heritability." While linkage analyses detect epistasis, defining the limits of the interacting segments poses a significant challenge especially when the interactions are between loci in close proximity. The goal of the present study was to isolate two such epistatic blood pressure (BP) loci on rat chromosome 5. A panel of S.LEW bicongenic strains along with the corresponding monocongenic strains was constructed. BP of each set comprising of one bicongenic and two corresponding monocongenic strains were determined along with the parental Salt-sensitive (S) strain. Epistasis was observed in one out of four sets of congenic strains, wherein systolic blood pressures (SBP) of the two monocongenic strains S.LEW(5)x6Bx9x5a and S.LEW(5)x6Bx9x5b were comparable to that of S, but the SBP of the bicongenic strain S.LEW(5)x6Bx9x5 (157 +/- 4.3 mmHg) was significantly lower than that of S (196 +/- 6.8 mmHg, P < 0.001). A two-way ANOVA indicated significant interactions between the LEW alleles at the two loci. The interacting loci were 2.02 Mb apart and located within genomic segments spanning 7.77 and 4.18 Mb containing 7,360 and 2,753 candidate variants, respectively. The current study demonstrates definitive evidence for epistasis and provides genetic tools for further dissection of the isolated epistatic BP loci.