ACTH stimulation is useful in assessing the hypothalamo-pituitary-adrenal axis. However, there is still some debate about the proper dose and interpretation. We designed a new protocol using repetitive graded ACTH stimulation. Thirty-two patients with the diagnosis of adrenal insufficiency (Al) were studied. After taking samples for baseline ACTH and cortisol, 1 microg fresh-prepared ACTH (Synacthen (1-24)) was injected intravenously, then 5, 50 and 100 microg at hourly interval. Cortisol responses were measured at 30, 60, 120, 180 and 240 min. The secondary Al group (26 subjects) had cortisol responses in between those of the control group (8 subjects) and the primary Al group (6 subjects). The minimal overlap between the secondary Al group and the control group occurred at a 30-min cortisol response after 1 microg ACTH stimulation, using 20 microg/dl as the cut-off level. There was only one exception which showed an episodic release at 30 min. There were 5, 10 and 9 patients with secondary Al who responded normally to 5, 50 and 100 microg ACTH stimulation, respectively. Maximal cortisol increments of the primary Al group were all below 4 microg/dl. Although there were 11 cases of secondary Al whose cortisol responses overlapped with those of primary Al, only two of them had a cortisol increment less than 4 microg/dl. Our new protocol combines the advantage of the low dose ACTH stimulation test, a sensitive method for detecting mild Al, and the ACTH infusion test, a longer test to mimic surgical stress.