In this study the effects of animal age combined with feeding regime and the utilisation of a beta-agonist (within a grain-fed system) on proximate composition and fatty acid profile of M. longissimus lumborum (LL), M. biceps femoris (BF), and M. semitendinosus (ST) were determined. Eighty Bonsmara steers consisting of A-age (0 permanent incisors) grain-fed (AC) and grain-fed supplemented with a beta-agonist, zilpaterol (AZ) (n = 20) grass-fed AB-age (1 – 2 permanent incisors; AB) (n = 20), and B-age (3 – 6 permanent incisors; B) (n = 20) animals were used. These four groups are representative of cattle slaughtered in South Africa and were treated as four production systems. The chemical composition of all three muscles showed that zilpaterol increased protein and reduced muscle fat contents of meat. All the muscles of both grass-fed groups (AB and B) had significantly higher content of certain desirable fatty acids (FAs) such as conjugated linoleic acid (CLA), omega-3 (n-3) FAs, branched chain saturated phytanic acid, and a lower omega-6/omega-3 (n-6/n-3) ratio than the two grain-fed groups (AC and AZ). The FA composition of grain-fed beef muscle was generally not influenced by the use of zilpaterol except for a tendency towards higher n-6 polyunsaturated fatty acid (PUFA) in beta-agonist produced beef. This was mainly due to higher levels of linoleic acid in LL and ST muscles and higher CLA in BF muscle of AZ animals. Phytanic acid was also higher in BF muscle of the AZ group compared to AC. Differences in animal age among grass-fed animals (AB vs. B) had minimal effect on FA composition of grass-fed beef. We can conclude that differences in FA composition of the three muscles are influenced mainly by feeding regime and less by differences in production factors within feeding regimes.