To examine the effect of fish oil supplementation on the fatty acid (FA) composition of human milk and maternal and infant erythrocytes, five lactating women were supplemented with 6 g of fish oil daily for 21 d. Usual maternal diets contained 1,147 mg of total n-3 FA, with 120 mg from very long-chain (> C18) n-3 FA. Supplementation increased dietary levels to 3,092 mg of total n-3 FA and 2,006 mg of very long-chain n-3 FA. Milk samples were collected daily, prior to fish oil ingestion, and at 4-h intervals on days 1, 7, 14 and 21. Milk n-3 FA content increased within 8 h and reached steady state levels within one week. The n-6 fatty acid content decreased. Erythrocyte eicosapentaenoic acid content increased from 0.24% to 1.4% (P < 0.01) in mothers and from 0.11% to 0.70% (P < 0.05) in infants. Docosapentaenoic acid increased from 1.4% to 2.2% (P < 0.05) in mothers and from 0.30% to 0.78% (P < 0.01) in infants. There was no significant change in docosahexaenoic acid or n-6 fatty acid content. Maternal platelet aggregation responses were variable. No differences in milk or plasma tocopherol levels were noted.