Within the Danish population, milk DHA (22:6n-3) levels vary by more than a factor of 10. This paper deals with fluctuations in the milk content of 22:6n-3 and other long-chain PUFA (LCPUFA) and the acute effects of fish meals and fish oil supplements on milk levels of LCPUFA. Twelve fish-eating mothers with 4-mon-old infants provided one blood and one adipose tissue sample, and seven consecutive morning hind-milk samples with dietary records from the previous days. Another 12 lactating women were given fish oil (2-8 g) for breakfast and delivered 6-12 milk samples during the following 24 h. The mean milk 22:6n-3 content of the fish-eating mothers was 0.57+/-0.28 FA% (= percentage of total area of FAME peaks in GLC) and the day-to-day variation (SD/mean) within the individual was 35+/-17%. Mean milk 22:6n-3 content on mornings with no fish the day before was 0.42+/-0.15 FA%; this was increased by 82+/-17% (n = 9, P = 0.05) if the mother had eaten fatty fish. Fish oil resulted in a twofold increase in milk 22:6n-3 levels, which peaked after 10 h and lasted for 24 h. The EPA content of milk was also increased by fish meals and fish oil supplements, but these had no effect on the level of arachidonic acid. The study showed that diurnal and day-to-day fluctuations in levels of milk n-3 LCPUFA are large, which makes it difficult to assess the 22:6n-3 intake of breast-fed infants from a single milk sample. In studies of the functional outcome of dietary 22:6n-3 in breast-fed infants it is suggested also to use a measure of maternal 22:6n-3 status.