This study evaluated the longitudinal effect of fish oil in pregnancy on breast milk fatty acid composition and infant outcomes. In a randomized, controlled trial, 98 women received 2.2 g docosahexaenoic acid (DHA) and 1.1 g eicosapentaenoic acid (EPA) or olive oil from 20 wk of gestation until delivery. Fatty acid composition in breast milk (at 3 d, 6 wk, and 6 mo) and infant erythrocyte membranes (at 1 y) were determined by gas liquid chromatography. Breast milk fatty acids were examined in relationship to growth and development. Compared with control group, breast milk from women who received fish oil had proportionally higher DHA and EPA levels at 3 d and 6 wk after delivery, but this difference was no longer apparent by 6 mo. Infant DHA status at 1 y of age was directly related to DHA levels at 3 d, 6 wk, and 6 mo postpartum (but not to antenatal supplementation). Both EPA and DHA in breast milk were positively correlated with Griffith's developmental scores including hand and eye coordination. Thus, supplementation in pregnancy was associated with increased n-3 long-chain polyunsaturated fatty acids (LCPUFAs) in breast milk, particularly in early lactation, and this was positively associated with infant DHA status at 1 y.