Evaluation of the HPA axis is still a challenge; due to different sensitivities and stimulation efficiencies of dynamic tests, lack of standard assays for cortisol measurement and lack of data regarding the effects of age and gender on the results of the HPA axis evaluation with different dynamic tests. This study was performed to compare 1 mu g ACTH, 250 mu g ACTH and glucagon tests in the evaluation of HPA axis. The study was carried out on 55 healthy individuals (28 men, 27 women). 10-12 volunteers were included from every decades between 20 and 70 years. Low dose short synacthen test (1 mu g ACTH), standard dose short synacthen test (250 mu g ACTH) and glucagon tests were performed consecutively. The mean peak cortisol response to standard dose ACTH stimulation test was found to be significantly higher than the low dose ACTH and glucagon stimulation tests. The mean peak cortisol responses to low dose ACTH and the glucagon stimulation tests were not significantly different. The mean peak cortisol responses did not differ significantly between different age or sex groups. The lowest peak cortisol responses obtained after low dose ACTH and glucagon stimulation tests were 12.5 and 9.1 mu g/dl respectively in the volunteers who all had cortisol responses higher than 20 mu g/dl after standard dose ACTH stimulation test. The lowest cortisol responses obtained during 250 mu g ACTH, 1 mu g ACTH and glucagon stimulation tests were found to be 20.1, 12.5 and 9.1 mu g/dl in a known group of healthy people. So the consideration of appropriate hormonal cut-off levels for each test seems reasonable. The age, sex and body mass indeces were not shown to affect the cortisol response to dynamic stimulation tests.