Epidemiologic studies on the protective role of omega-3 fatty acids (n:3) on breast cancer prevention remain inconclusive but studies in preclinical models provide more positive outcome. However, the mechanisms accounting for the protective effect of n:3 are not defined. In the present study, conducted in the N-methyl-N-nitrosourea-induced rat mammary carcinogenesis model, we examined the effects of n:3 individually and in combination with the anti-estrogen Tamoxifen (Tam) on a comprehensive panel of systemic and preneoplastic mammary gland restricted biomarkers which may be critical in the progression to invasive cancer. We observed that fish oil (FO) rich diets significantly reduced Ki67 expression in hyperplastic lesions, while cleaved caspase-3 expression was not affected. Dietary FO and/or Tam did not have major effects on systemic oxidative stress biomarkers, based on oxidative damage to DNA measured as 8-hydroxy-2-deoxyguanosine (8-OH-dG) and lipid peroxidation assessed as thiobarbituric acid reactive substances (TBARS). Tissue levels of 8-isoprostane, on the other hand, were markedly reduced (p<0.0001) in FO-fed rats, possibly as a result of FO-induced depletion of arachidonic acid in the mammary gland. These results suggest that the protective effect of n:3 in this experimental system is not mediated by changes in the levels of oxidative stress but may result from suppression of arachidonic acid-specific pathways.