Objective—To determine whether trilostane or ketotrilostane is more potent in dogs and determine the trilostane and ketotrilostane concentrations that inhibit adrenal gland cortisol, corticosterone, and aldosterone secretion by 50%. Sample—24 adrenal glands from 18 mixed-breed dogs. Procedures—Adrenal gland tissues were sliced, placed in tissue culture, and stimulated with 100 pg of ACTH/mL alone or with 5 concentrations of trilostane or ketotrilostane. Trials were performed independently 4 times. In each trial, 6 samples (1 for each time point) were collected for each of the 5 concentrations of trilostane and ketotrilostane tested as well as a single negative control samples. At the end of 0, 1, 2, 3, 5, and 7 hours, tubes were harvested and media and tissue slices were assayed for cortisol, corticosterone, aldosterone, and potassium concentrations. Data were analyzed via pharmacodynamic modeling. One adrenal slice exposed to each concentration of trilostane or ketotrilostane was submitted for histologic examination to assess tissue viability. Results—Ketotrilostane was 4.9 and 2.4 times as potent in inhibiting cortisol and corticosterone secretion, respectively, as its parent compound trilostane. For trilostane and ketotrilostane, the concentrations that inhibited secretion of cortisol or corticosterone secretion by 50% were 480 and 98.4 ng/mL, respectively, and 95.0 and 39.6 ng/mL, respectively. Conclusions and Clinical Relevance—Ketotrilostane was more potent than trilostane with respect to inhibition of cortisol and corticosterone secretion. The data should be useful in developing future studies to evaluate in vivo serum concentrations of trilostane and ketotrilostane for efficacy in the treatment of hyperadrenocorticism.