Fatty acid (FA) composition of intramuscular fat (IMF) in M. Longissimus dorsi (LD) was measured in 72 steers from Angus (A), Charolais×Angus (CHA×A) and Holstein Argentine (HA) breeds. The steers were allotted to four dietary treatments of six animals each: T1, steers grazed on pasture; T2, steers supplemented with cracked corn grain (0.7% of live-weight) daily and free access to pasture; T3, steers supplemented with cracked corn grain (1% of live-weight) daily and free access to pasture; and T4, feedlot (concentrate based on corn, alfalfa hay and soybean meal without access to pasture). At slaughter weight, samples of LD at the 11th rib were used for intramuscular lipid analysis. The diet was shown to be more important than breed in determining FA composition. Pasture beef had higher percentages of saturated fatty acids (SFA), n-3 polyunsaturated fatty acids (PUFA) and conjugated linoleic acid (CLA) and lower percentages of IMF, monounsaturated fatty acids (MUFA), n-6 PUFA and n-6/n-3 ratios than feedlot beef. HA beef presented lower percentages of SFA and more MUFA with a higher n-6/n-3 ratio than A and CHA×A. Comparing grass and feedlot beef the amounts of FA in muscle (mg/100g) were, respectively 18:3 n-3 (44 vs. 11mg), CLA (20 vs. 12mg), 20:5 n-3 (20 vs. 11mg), 22:5 n-5 (20 vs. 11mg), 22:6 n-3 (12 vs. 6mg) and n-3 PUFA (84 vs. 32mg). Feedlot beef has more SFA (1372 vs. 1081mg), MUFA (1574 vs. 1078mg), PUFA (350 vs. 227mg) and n-6 PUFA (318 vs.143mg).