The effects of short-term phenobarbital administration were evaluated in 6 adult mixed-breed dogs that received phenobarbital (5 mg/kg of body weight, PO, q 12 h) for 8 consecutive weeks. Six additional dogs served as untreated controls. At 2-week intervals, endogenous adrenocorticotropic hormone (ACTH) concentration and cortisol concentration before and 2 hours after administration of porcine aqueous ACTH (2.2 IU/kg, IM) were measured. By means of one-way ANOVA, we were not able to detect a significant (P > or = 0.05) difference in endogenous ACTH concentration and cortisol concentration before and after exogenous ACTH administration within groups over time or between groups at any time. To evaluate effects of long-term phenobarbital administration, sera and plasma were collected from 5 epileptic dogs that had received phenobarbital for > 2 years and had serum phenobarbital concentrations > 20 micrograms/dl. Endogenous ACTH concentration and cortisol concentration, before and after administration of ACTH, were within established reference ranges for all 5 dogs. Together, these results suggest that phenobarbital administration alone does not affect endogenous ACTH concentration or response to exogenous ACTH administration in dogs, and that these may be valid screening tests for hyperadrenocorticism in most dogs receiving phenobarbital.