We have determined the free cortisol concentration in serum using either the Amicon MPS-1 ultrafiltration-centrifugation method (I) or equilibrium dialysis (II). If procedure I was used we found that [1,2,6,7-3H]-, and [4-14C]cortisol had a lower affinity than unlabelled cortisol for corticosteroid binding globulin (CBG). The binding affinity (Ka) to three separate CBG-containing samples was 8-18 times lower for [1,2,6,7-3H]cortisol and 30-90 times lower for [4-14C]cortisol, when compared with that of unlabelled cortisol. This difference in affinity to CBG was not observed if method II was used for the free cortisol determinations. The observed isotope effect in method I is not caused by unspecific binding to material such as the Amicon MPS-1 chamber or to impurities in the tracer. We suggest that the centrifugation step during ultrafiltration changed the conformation of CBG, thereby reducing its affinity for labelled cortisol. It is concluded that incorrect results will be obtained if radiolabelled is cortisol used for determining the free cortisol content of plasma with the Amicon MPS-1 device.