n-3 Fatty acids reduce the risk of cardiovascular disease via a number of possible mechanisms. Despite this, there has been concern that these fatty acids may increase lipid peroxidation. The data in vivo are inconclusive, due in part to limitations in the methodologies. In this regard, the measurement of F2-isoprostanes provides a reliable assessment of in vivo lipid peroxidation and oxidant stress. This study aimed to assess the effects of supplementation with purified eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), the two major n-3 fatty acids, on urinary F2-isoprostanes and markers of inflammation, in type 2 diabetic patients. In a double-blind, placebo controlled trial of parallel design, 59 nonsmoking, treated-hypertensive, type 2 diabetic subjects, were randomized to 4 g daily of purified EPA, DHA, or olive oil for 6 weeks, while maintaining their usual diet. F2-isoprostanes, measured using gas chromatography-mass spectrometry in 24 h urines and C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), were measured before and after intervention. Thirty-nine men and 12 women aged 61.2 ± 1.2 years, with body mass index (BMI), 29.5 ± 0.5 kg/m2; 24 h blood pressure, 138/73 mmHg; HbA1c, 7.3 ± 0.1% and fasting glucose, 7.9 ± 0.2 mmol/l completed the intervention. Baseline urinary F2-isoprostanes were positively associated with HbA1c (p = .011) and fasting glucose (p = .032). Relative to the olive oil group, postintervention urinary F2-isoprostanes were decreased 19% by EPA (p = .017) and 20% by DHA (p = .014). There were no significant changes in CRP, IL-6, and TNF-α following EPA or DHA supplementation. In regression analysis, Δ F2-isoprostanes were positively associated with Δ HbA1c (p = .007) independent of treatment group; and with Δ TNF-α (p = .034) independent of age, gender, BMI, and treatment group. There were no associations with Δ CRP or Δ IL-6. This study is the first report demonstrating that either EPA or DHA reduce in vivo oxidant stress without changing markers of inflammation, in treated hypertensive, type 2 diabetic subjects.