Fourteen crossbred beef steers (average initial BW 313 ± 13.1 kg) fed a 90% concentrate diet (as-fed basis) were used to evaluate effects of ionophores on serum metabolic hormones and clinical chemistry profiles. Treatments were no ionophore (C; four steers), lasalocid (L; 33 mg/kg of diet; five steers), and monensin (33 mg/kg of diet) plus tylosin (11 mg/kg of diet; MT; five steers). All steers were adapted to the 90% concentrate diet, after which treatments were applied and blood was sampled via jugular catheters on d 7, 35, 63, 91, and 119 of the trial at 0, 1, 2, 3, 4, 6, 8, and 10 h after feeding. Averaged over sampling days, serum glucose concentrations did not differ (P >.10) among treatments at any sampling time (treatment × sampling time, P < .05). Averaged across sampling days and times, serum growth hormone, insulin, cholesterol, and triglyceride concentrations did not differ (P >.10) among treatments. No major effects of ionophores were noted for serum enzymes or protein fractions. Averaged over sampling times within day (treatment × day interaction, P < .10), both L and MT increased (P < .05) serum Ca and Na concentrations on d 91 and 119 compared with C. Serum inorganic P was increased (P < .10) for L vs MT and for ionophore treatments compared with C (P < .10) on several occasions (treatment × day × sampling time interaction, P < .05). Results suggest that ionophores do not cause dramatic changes in serum metabolic hormones or clinical chemistry profiles; however, monensin and lasalocid altered serum minerals in beef steers fed a high-concentrate diet.