The common practice in North American feedlot industries is to add antibiotics to the diet to prevent disease and improve both BW gain and feed efficiency. In this study, 240 crossbred steer calves were backgrounded on a 54% silage diet for 80 d and fed a finishing diet consisting of 81% barley grain, 10% barley silage, and 7.5% supplement (DM basis) with and without in-feed antibiotics for approximately 120 d. Calves were assigned to 1 of 5 treatments: a control with no antibiotics, 11 mg/kg of chlortetracycline, 44 mg/kg of chlortetracycline, 44 mg/kg of chlortetracycline plus 44 mg/kg of sulfamethazine, and 11 mg/ kg of tylosin phosphate. A combination of GLC and silver-ion HPLC methods was used to analyze the fatty acid composition of brisket adipose tissue, with emphasis on trans-18:1 and CLA isomers. The inclusion of nonionophore antibiotics in the diet had little effect on the fatty acid composition, except that feeding either 44 mg/kg of chlortetracycline or 11 mg/kg of tylosin caused small increases in 9c-14:1 and 16:0 relative to the control (0.26 and 0.9 g/100 g of total fatty acids, respectively). Likewise, profiles of trans-18:1 and CLA isomers were unchanged by antibiotics, but across treatments the predominant trans-18:1 isomer was 10t-18:1 (where t = trans; 3.22%) at 3 times the concentration of the second most abundant isomer (11t-18:1; vaccenic acid, 1.05%). Rumenic acid (9c,11t-18:2, where c = cis) was the major CLA isomer at 61% of total CLA, followed by 7t,9c-18:2 at 9%. Because no other effects on fatty acid composition were evident, data for trans-18:1 and CLA were pooled across treatments to investigate possible relationships among rumen PUFA metabolites. The total trans-18:1 content in brisket adipose tissue was positively correlated with 10t-18:1, but not with 11t-18:1, whereas the total CLA was positively correlated with 9c,11t-18:2, but not with 7t,9c-18:2. The 7t,9c-18:2 was, however, positively correlated with 10t-18:1 and 6t/7t/8t-18:1 but was negatively correlated with rumenic acid. These metabolic interrelationships suggest the presence of bacterial populations with distinct pathways for PUFA biohydrogenation in which either 10t-18:1 or 11t-18:1 predominate. Overall, the nonionophore antibiotics tested did not appreciably change adipose tissue composition and consequently could not be used to improve the trans-18:1 or CLA profile (i.e., increase vaccenic and rumenic acids at the expense of 10t-18:1).