Technical Abstract: Substantial evidence relates increased sex hormone concentrations with increased breast cancer risk. Varying omega-3 fatty acid (n-3) intake may lead to alterations in eicosanoid balance and subsequent changes in circulating sex hormones that reduce risk. To clarify effects of dietary fat and n-3 intake on breast cancer risk markers, circulating sex hormones and urinary eicosanoids were measured in response to controlled feeding of diets designed to increase plasma concentrations of n-3. A controlled cross-over feeding trial in 17 postmenopausal women was conducted using three diets: high fat (HF; 40% energy from fat), low fat (LF; 20% energy from fat) and low fat plus n-3 (LFn3; 20% of energy from fat plus 3% of energy from n-3) in 8-wk feeding periods. Plasma phospholipid fatty acids (PLFA), plasma sex hormones and urinary eicosanoids were measured in response to each diet. Plasma PLFA n-3 were increased with the LFn3 relative to HF and LF (P < 0.0001). Urinary prostaglandin E metabolite (PGE-M) increased with HF relative to LF and LFn3 (P = 0.02) and urinary 11-dehydro-thromboxane B2 (TxB-M) increased with HF (P = 0.01). Plasma estradiol (E2) was increased by HF (P = 0.03) and plasma follicle stimulating hormone (FSH) was increased by LF (P = 0.03). There was a trend for reduced estrone with LFn3 relative to HF and LF (P = 0.14). These results support a role of low fat diet and n-3 in reduction of breast cancer risk relative to a high fat diet.