BACKGROUND For diagnostic tests used in the evaluation of patients with Cushing's syndrome, well defined reference values were lacking in our laboratory. In the present study, we established reference values based upon test results of 50 subjects recruited from the general population. METHODS We studied 50 subjects not suspected of having Cushing's syndrome, equally distributed according to sex and age between 20 and 69 years. In addition to 24 h urinary excretion of free cortisol for two days, a low-dose (1 mg) overnight dexamethasone suppression test, a corticotropin releasing hormone stimulation test (CRH test) and a high-dose (7 mg) intravenous dexamethasone suppression test were performed. Reference values are given as the observed range. RESULTS There was considerable intra-subject variation in 24 h urinary cortisol excretion which could not be merely attributed to incompleteness of the urine collection. The following reference values were established: 24 h urinary free cortisol excretion: 15-145 nmol/24 h; overnight dexamethasone suppression test: cortisol on day 2 < 50-230 nmol/l; CRH test: cortisol increase 15-289%, ACTH increase 36-12.100%; high-dose, intravenous dexamethasone suppression test: cortisol after 7 h < 50-97 nmol/l; cortisol after 24 h < 50-50 nmol/l. There were no significant effects of age on any of the parameters studied. Apart from higher basal ACTH plasma concentrations in men, no sex differences were observed. CONCLUSIONS We established reference values for tests that can be useful in the evaluation of patients with possible Cushing's syndrome.