We examined the ability of n-3 FA in flaxseed-supplemented rations to increase the n-3 FA content of bovine muscle. Two groups of animals were used in each of two separate trials: (i) Hereford steers supplemented (or not) with ground flaxseed (907 g/d) for 71 d, and (ii) Angus steers supplemented (or not) with ground flaxseed (454 g/d for 3 d followed by 907 g/d for 110 d). For the Hereford group, flaxseed-supplemented rations increased 18:3n-3 (4.0-fold), 20:5n-3 (1.4-fold), and 22:5n-3 (1.3-fold) mass as compared with the control, and increased total n-3 mass about 1.7-fold. When these data were expressed as mol%, the increase in 18:3n-3 was 3.3-fold and in 20:5n-3 was 1.3-fold in the phospholipid fraction, and 18:3n-3 was increased 4-fold in the neutral lipid fraction. For the Angus group, flaxseed ingestion increased masses and composition of n-3 FA similarly to that for the Herefords and doubled the total n-3 FA mass. The effect of cooking to a common degree of doneness on FA composition was determined using steaks from a third group of cattle, which were Angus steers. We demonstrated no adverse effects on FA composition by grilling steaks to an internal temperature of 64 degrees C. Because n-3 FA may affect gene expression, we used quantitative real-time reverse transcriptase-polymerase chain reaction to quantify the effect of feeding flaxseed on heart-FA binding protein, peroxisome proliferator activated receptor gamma (PPARgamma) and alpha (PPARalpha) gene expression in the muscle tissue. PPARgamma mRNA level was increased 2.7-fold in the flaxseed-fed Angus steers compared with the control. Thus, we demonstrate a significant increase in n-3 FA levels in bovine muscle from cattle fed rations supplemented with flaxseed and increased expression of genes that regulate lipid metabolism.