Based on potential health benefits, there is a need to develop effective strategies for enhancing milk fat concentrations of cis-9 18[ratio ]1, 18[ratio ]3 n-3 and conjugated linoleic (CLA) content in milk without compromising the sensory or storage characteristics of processed milk or dairy products. Sixteen Finnish Ayrshire dairy cows were used in a cyclic change-over experiment with four 21-d experimental periods and a 4×2×2 factorial arrangement of treatments to evaluate the effects of forage conservation method, concentrate level and supplements of propylene glycol (PG), and their interactions on milk fatty acid composition and vitamin content. Experimental treatments consisted of four conserved forages offered ad libitum, supplemented with two levels of a standard concentrate (7 or 10 kg/d) and PG (0 and 210 g/d) fed as three equal meals. Primary growths of timothy and meadow fescue sward were conserved by ensiling with none (NA), an inoculant enzyme preparation (IE) or a formic acid based (FORM) additive or as hay 1 week later. Conservation of grass by drying rather than ensiling resulted in lower forage 18[ratio ]2n-6, 18[ratio ]3n-3, total fatty acid and fat-soluble vitamin concentrations. In spite of lower intakes, milk fat 18[ratio ]2n-6 and 18[ratio ]3n-3 content was higher (P<0·05) for hay than for silage diets (12·1, 9·6, 9·6 and 9·3 and 5·00, 3·51, 4·27 and 2·93 g/kg total fatty acids, for hay, NA, IE and FORM silages, respectively). Forage conservation method had no clear effects on milk trans 18[ratio ]1 or CLA content. Compared with silage, hay diets resulted in milk containing lower (P<0·001) riboflavin, α-tocopherol and β-carotene concentrations, but had no effect on ascorbic acid, thiamine, pyridoxine or retinol content. Feeding more concentrates had no effect on milk fatty acid composition or milk vitamin content, other than lowering (P<0·001) 16[ratio ]0 concentrations from 348 to 338 g/kg fatty acids. Supplements of PG led to small (P<0·05) increases in milk 13[ratio ]0 anteiso and 15[ratio ]0 content from 1·06 and 11·3 to 1·22 and 12·6 g/kg fatty acids and reduced (P<0·05) the concentrations of ascorbic acid (16·1 v. 15·1 g/kg milk).