Effect of testing environment on adrenal cortical responses to an injection of ACTH in clinically normal dogs was examined in three locations, presumably of increasing order of stress elicitation: in a home; veterinary hospital (VH), 4 hours in a cage; and VH, overnight in a cage. Basal cortisol (hydrocortisone) values for plasma were significantly lower (P less than 0.001) for the home group (1.8 microgram/dl) when compared with values for the VH, 4-hour cage (3.8 microgram/dl) or the VH, overnight cage (3.9 microgram/dl) groups. However, significant differences (P greater than 0.05) were not observed 2 hours after ACTH admininstration for the home group (13.7 microgram/dl); VH, 4-hour cage group (14.8 microgram/dl); or VH, overnight cage group (16.0 microgram/dl). Responses of individual dogs were consistent (P less than 0.005). The testing environment did not markedly affect results of adrenal cortical function tests for dogs when ACTH stimulation was utilized. The response of dogs to ACTH, as monitored by immunologic assay techniques (competitive protein-binding assay or radioimmunoassay), was consistent and was useful as a diagnostic aid for adrenal malfunction.