We investigated the fatty acid composition of the phospholipid fraction of the shoulder muscle (triceps brachii) from Jersey and Limousin yearling steers, yearling heifers, and nonlactating cows. The aim was to study breed, sex, and age differences. Significant breed differences in some individual fatty acids were apparent between Jersey and Limousin cows. Limousin cows had more palmitate, vaccenate, arachidonate, and less gamma-linolenate and eicosapentanoate than Jersey cows. Age differences were significant: proportions of palmitate, stearate, and oleate decreased and linoleate, arachidonate, and total polyunsaturates increased with age. Most of the breed x age interactions were not significant. Also, phospholipids of Jersey and Limousin cows did not differ in total saturated, monounsaturated, and polyunsaturated fatty acids. Yearling data revealed significant sex differences in most of the fatty acids, including total monounsaturates and polyunsaturates. Yearling steers had more myristate, palmitoleate, stearate, and total monounsaturates and less linoleate, arachidonate, eicosapentanoate, and total polyunsaturates than heifers. Breed differences were also significant: Limousin yearlings had more di-homogamma-linolenate and erucate and less eicosapentanoate and nervonate than their Jersey counterparts. The sex x breed interaction was not significant for most of the fatty acids. These results imply that breed, age, and sex are important factors that influence the fatty acid composition of muscle phospholipids in cattle.