The dose of 250 microg used in the standard short synacthen test is supraphysiological and lower doses may provide a more sensitive test. We examined steroid responses to 125ng/m2, 250ng/m2 and 500 ng/m2 (1-24)ACTH in 6 normal males, looking at effects of dose and the within- and between-subject coefficients of variation (CV). Subjects were given each dose 3 times, blood samples were taken at 10 minute intervals. There was a dose response relationship between dose of (1-24)ACTH and peak values for cortisol and 17OHP (p<0.05). There was no difference between peaks of A4 at different doses and no clear peaks were reached for DHEAS. 86% of the peaks for 17OHP, 63% for A4 and 25% for cortisol were at 10 minutes and 14%, 29% and 65% respectively at 20 mins (p=0.001). Within-subject CV for cortisol was 12.6% and between subject 10.1%. Tests of adrenal function using low doses of (1-24)ACTH have acceptable between- and within-subject CV for peak values with a dose as low as 125 ng/m2 (1-24)ACTH. Protocols for low dose synacthen tests, with traditional sampling at zero, 30 and 60 minutes or even as shown here at 10 minute intervals, fail to fully define the changes in steroid levels following adrenal stimulation. More frequent blood sampling will be needed to accurately detect peak levels in particular of 17OHP and A4.