Phenobarbital can interfere with the thyroid axis in human beings and rats by accelerating hepatic thyroxine metabolism because of enzyme induction. In human beings, it also can interfere with the low-dose dexamethasone suppression test (LDDST) used to assess adrenal function by accelerating dexamethasone metabolism. This effect can cause a lack of suppression of pituitary ACTH and subsequent adrenal cortisol release after dexamethasone administration. The effects of phenobarbital on the thyroid axis, the adrenal axis, and adrenal function tests were prospectively investigated in 12 normal, adult dogs. Phenobarbital was administered at 5 mg per kilogram of body weight (range, 4.8-6.6 mg/kg) PO q12h for 29 weeks, resulting in therapeutic serum concentrations (20-40 microg/mL). Serum total thyroxine (TT4), free thyroxine (FT4) by equilibrium dialysis, total triiodothyronine (TT3), thyrotropin (TSH), and cholesterol were determined before and during phenobarbital treatment. LDDST, ACTH stimulation tests, and ultrasonographic evaluation of the adrenal glands were performed before and during treatment. TT4 and FT4 decreased significantly (P < or = .05), TT3 had minimal fluctuation, TSH had only a delayed compensatory increase, and cholesterol increased during phenobarbital treatment. The delayed increase in TSH, despite persistent hypothyroxinemia, suggests that accelerated hepatic thyroxine elimination may not be the only effect of phenobarbital on the thyroid axis. There was no significant effect of phenobarbital on either of the adrenal function tests. With the methods employed, we did not find any effects of the drug on the hormonal equilibrium of the adrenal axis.