Our objective was to determine the effect of breed (B) and slaughter age/weight on the fatty acid composition, particularly the conjugated linoleic acid (CLA) concentration of beef. Two strains of a dairy breed [Holstein-Friesian (HF) (n=16 steers/strain)] and a late-maturing breed [Belgian Blue×Holstein-Friesian (BB) (n=16 steers)] were used. The HF strains were either of New Zealand (NZ) or European-American (EU) descent (selected in a grazed grass or a high concentrate nutritional environment, respectively). Animals were grown from calves to either a light (L; 560kg) or heavy (H; 630kg) target slaughter weight. Samples of M. longissimus dorsi were collected post-slaughter, lipids were separated into neutral (NL) and polar (PL) fractions, and fatty acid composition determined by gas-chromatography. The total fatty acid concentration and the concentrations of cis9, trans11 CLA, total CLA, MUFA and SFA in total intramuscular lipids were lower and the P:S ratio higher for BB than NZ or EU which did not differ. These differences largely reflected the changes in NL. The C18 desaturase index was higher for NZ than EU but EU did not differ from BB. Increasing slaughter weight/age increased the total fatty acid concentration and the concentrations cis9, trans11 CLA (P=0.06), total CLA (P=0.06), trans11 C18:1, MUFA and SFA and the C18 desaturase index and decreased the P:S ratio. The n-6:n-3 PUFA ratio was similar at the lighter slaughter weight/age for the three breeds/strains but increased for NZ and EU with increasing slaughter weight/age but was not affected in BB. It is concluded that the CLA concentration largely reflected muscle fatness but that increasing slaughter weight/age differentially affected the n-6:n-3 PUFA ratio of beef from early and late-maturing cattle.