We developed a time-resolved-fluoro-immunoassay to measure cortisol binding globulin (CBG) in serum. It is a microtitre plate, solid phase, reagent excess, sandwich assay in which the same polyclonal antiserum is used as a source of capture and labeled antibodies. The results of this assay were shown to be reliable and were fully comparable with those obtained by a commercially available kit. As a reflection of the free cortisol concentration we measured cortisol and CBG concentrations in serum and calculated the Free Cortisol Index (FCI) = [cortisol]serum/[CBG]serum.100. The clinical use of this parameter, as a screening test for disturbances of the pituitary-adrenal axis, was investigated in different groups of subjects: healthy men and women, women using oral contraceptives, pregnant women at term, patients with thyroidal illnesses, patients using anti-epileptic drugs and patients suffering from adrenal diseases. In a number of groups we compared the FCI results with measurements of cortisol in saliva, another parameter used as an estimate of the concentration of free cortisol in blood. Our conclusion is that the FCI, in contrast to a total cortisol measurement alone, can prevent unnecessary further testing.