Because of the potential serious side effects associated with mitotane use, trilostane has largely replaced mitotane for treatment of spontaneous canine hypercortisolism. However, trilostane does not affect growth or metastases of cortisol-secreting adrenocortical tumors, which account for 15% to 20% of cases. New targeted treatment options are therefore desirable. This study first showed that a canine primary adrenocortical cell culture can be a useful in vitro system for testing new treatment options. Based on the importance of steroidogenic factor-1 (SF-1) on adrenal steroidogenesis, the study then examined the effect of 3 SF-1 inverse agonists on cortisol production using this in vitro culture system. One of the tested compounds, #31, was shown to be an effective inhibitor of cortisol production and SF-1 target gene expression in non-ACTH–stimulated and ACTH-stimulated normal adrenocortical cells, warranting further study of its use as a potential treatment option in canine hypercortisolism.