The serum concentrations of cortisol and cortisone were measured in 19 healthy dogs and in 13 dogs with pituitary-dependent hyperadrenocorticism (pdh) before and one hour after an injection of synthetic adrenocorticotropic hormone (acth). In the dogs with pdh, the cortisol and cortisone concentrations were measured before and after one to two weeks and three to seven weeks of treatment with trilostane. The dogs with pdh had significantly higher baseline and poststimulation concentrations of cortisol and cortisone, and higher baseline cortisol:cortisone ratios than the healthy dogs. During the treatment with trilostane, the poststimulation cortisol, the baseline and poststimulation cortisone concentrations, and the baseline and poststimulation cortisol:cortisone ratios decreased significantly. The decrease in poststimulation cortisone was significantly smaller than the decrease in cortisol.