Information on lipids in forages is scarce, and effects of nitrogen (N) application level and regrowth period on the fatty acid (FA) concentration and composition of perennial ryegrass (Lolium perenne L.) were studied. N was applied at 0, 45 and 100 kg ha 21 , and swards were cut after various regrowth periods, resulting in six treatments designed as randomised blocks with three replicates. The stages of development ranged from vegetative to elongating swards, with herbage yield levels from 1.9 to 4.2 t dry matter (DM) ha 21 . Concentrations of individual FA were determined by gas chromatography, and canopy characteristics and herbage quality were assessed. The treatments resulted in canopies with contrasting DM yields and protein concentrations. Five FAs, representing 98% of total FAs, were studied in detail. On an average, the concentration of these major FAs in fresh grass was 15.1 g kg 21 DM, and 69% of the major FAs consisted of C18:3. Regrowth period affected (P < 0.05) the total FA concentration, and significantly (P < 0.01) lower concentrations of C18:3 and C16:1 were found after a longer period of regrowth. N application resulted in higher (P < 0.001) concentrations of all FAs. The FA composition was not affected by N application, but a longer regrowth period significantly (P < 0.001) decreased the proportion of C18:3 and increased those of C18:2 and C16:0. A strong, positive overall linear relation was found between the concentrations of total FAs and C18:3 with the crude protein concentration in the herbage. These studies demonstrate opportunities to affect the FA concentration and composition of FA in herbage through management strategies, which could affect milk FA composition.