The aim of this research was to investigate the effect of fish oil supplementation, in the third trimester of pregnancy and early lactation period of healthy pregnant Danish women. Forty-four pregnant women were randomly allocated to fish oil supplementation (1.3 g EPA and 0.9 g DHA per day) from week 30 of gestation (FO-group) or to a control regimen (olive oil or no oil; controls). The FO-group was randomly subdivided into women stopping fish oil supplementation at delivery [FO(pregn)], and women continuing supplementation for an additional 30 d [FO(pregn/lact)]. Thirty-six women agreed to collect milk samples at days 4, 16, and 30 postpartum. The FA composition of the milk samples was determined by GLC. At days 4, 16, and 30 in lactation, FO(pregn/lact) women (n = 12) had, respectively 2.3 (P = 0.001), 4.1 (P = 0.001), and 3.3 (P = 0.001) times higher mean contents of LCPUFA(n-3) in their breast milk compared with controls (n = 13), and 1.7 (P = 0.005), 2.8 (P = 0.001), and 2.8 (P = 0.001) times higher LCPUFA(n-3) contents, respectively, at these days compared with FO(pregn) women (n = 11). The latter group did not differ significantly from controls with regard to LCPUFA(n-3) content in the breast milk. Similar results were obtained when analyzing separately for effects on the milk content of DHA. Dietary supplementation with 2.7 g LCPUFA(n-3) per day from week 30 of gestation and onward more than tripled the LCPUFA(n-3) content in early breast milk; supplementation limited to pregnancy only was much less effective.