When forages represent a high proportion of ruminant diets they provide a significant quantity of fatty acids (FA). Effects of growth stage, fertilization, conservation method, growth period, species, and cultivar on forage FA were determined in four experiments. Concentrations of C16:0, C18:2, C18:3, and total FA (TFA) in timothy (Phleum pratense L.) decreased (P < 0.01), respectively, by 15, 16, 31, and 23% between stem elongation and early flowering. Nitrogen fertilization (120 vs. 0 kg N ha(-1)) caused an increase (P < 0.01) of 18% of C16:0, 12% of C18:2, 40% of C18:3, and 26% of TFA concentrations. Phosphorus was not deficient and P fertilization (45 vs. 0 kg P ha(-1)) had no significant effect on timothy FA concentrations. Wilting and drying decreased (P < 0.01) timothy C18:2, C18:3, and TFA concentrations. Concentrations of C18:2, C18:3, and TFA were higher in summer regrowth than in spring growth, primarily in orchardgrass (Dactylis glomerata L.) and timothy (P < 0.01). Significant variation for all FA concentrations was observed among 12 species (P < 0.05); on average, the C 18:3, C 18:2, and C 16:0 accounted for 88% of TFA in studied species. Timothy was the only species in which the difference among cultivars was simultaneously significant (P < 0.05) for concentrations of C18:2, C18:3, and TFA. Among the grasses, an annual ryegrass (Lolium multiflorum Lam.) cultivar had the highest C 18:3 concentration (20.6 mg g(-1) DM) whereas a timothy cultivar had the lowest (7.3 mg g(-1) DM) (P < 0.05). Among legumes, a white clover (Trifolium repens L.) cultivar had the highest C18:3 concentration (16.5 mg g(-1) DM) whereas an alfalfa (Medicago sativa L.) cultivar had the lowest (6.0 mg g(-1) DM) (P < 0.05). Polyunsaturated FA concentrations in forages can be increased by harvesting timothy at an early stage of development and as fresh grass, by increasing N fertilization of timothy, and by choosing species with higher FA concentrations such as white clover and annual ryegrass.