The mechanisms that can account for the remarkable mammary carcinogenicity of the environmental pollutant 6-nitrochrysene (6-NC) in the rat remain elusive. In our previous studies, we identified several 6-NC-derived DNA adducts in the rat mammary gland; one major adduct was derived from (±)-trans-1,2-dihydroxy-1,2-dihydro-6-nitrochrysene (1, 2-DHD-6-NC). In the present study, we resolved the racemic (±)-1, 2-DHD-6-NC into (−)-[R,R]- and (+)-[S,S] -1,2-DHD-6-NC and compared their in vivo mutagenicity and carcinogenicity in the mammary glands of female transgenic (BigBlue F344 × Sprague-Dawley)F1 rats harboring lacI/cII and Sprague-Dawley rats, respectively. Both [R,R]- and [S,S]-isomers exerted similar mutagenicity and carcinogenicity but were less potent than 6-NC. Additional in vivo and in vitro studies were then performed to explore possible mechanisms that can explain the higher potency of 6-NC than 1, 2-DHD-6-NC. Using ELISA, we found that neither 6-NC nor 1, 2-DHD-6-NC increased the levels of several inflammatory cytokines in plasma obtained from rats 24 h after treatment. In MCF-7 cells, as determined by immunoblotting, the effects of 6-NC and 1, 2-DHD-6-NC on protein expression (p53, Akt, p38, JNK, c-myc, bcl-2, PCNA, and ERβ) were comparable; however, the expressions of AhR and ERα proteins were decreased by 6-NC but not 1, 2-DHD-6-NC. The expression of both receptors was decreased in mammary tissues of rats treated with 6-NC. Our findings suggest that the differential effects of 6-NC and 1, 2-DHD-6-NC on AhR and ERα could potentially account for the higher carcinogenicity of 6-NC in the rat mammary gland.