A prospective study was undertaken to compare intravenous tetracosactrin at doses of 5 microg/kg and 250 microg for diagnosing hyperadrenocorticism in dogs. Both healthy dogs and dogs with pituitary-dependent hyperadrenocorticism were evaluated with the two doses of the drug, and serum cortisol concentrations were compared at 60 minutes post-stimulation. Some of the dogs had additional samples taken at 90 and 120 minutes. For four dogs with hyperadrenocorticism, timed samples were also obtained at 150, 180 and 240 minutes post-injection. Cortisol concentrations 60 minutes after stimulation with either 5 microg/kg or 250 microg intravenous tetracosactrin were similar for both healthy dogs and dogs with hyperadrenocorticism. The lower dose can therefore be used for diagnosing hyperadrenocorticism in dogs.