In severely ill patients low concentrations of the corticosteroid binding globulin are typically found; the aim of this study was to quantify directly free bioactive cortisol concentrations in the sera of postoperative cardiosurgical patients. Serum samples of 12 consecutive patients undergoing aortocoronary bypass surgery taken preoperatively and on the postoperative days 1 to 4 were analyzed. Total serum cortisol was quantified using liquid chromatographytandem mass spectrometry with an online sample extraction system and trideuterated cortisol as the internal standard, and free serum cortisol was measured after overnight equilibrium dialysis. Whereas on the first postoperative day, the median total serum cortisol concentration was approximately twofold increased compared to preoperative samples (preoperatively, 245 nmol/l (interquartile range (IQR) 203293 nmol/l); first postoperative day, 512 nmol/l (IQR 410611 nmol/l)), median dialyzable free cortisol concentration was almost sevenfold increased (preoperatively, 14.2 nmol/l (IQR 10.920.7 nmol/l); first postoperative day, 98.3 nmol/l (IQR 81.3134 nmol/l)). On the fourth postoperative day, median free cortisol was still significantly increased compared to baseline sampling (p < 0.05), whereas median total cortisol was not. A median of 5.7% (IQR 5.47.0%) of total cortisol was found as free cortisol on the preoperative day, 21.2% (IQR 18.9 23.5%) on the first postoperative day and 10.5% (IQR 9.814.0%) on the fourth postoperative day. It is concluded that during the postoperative period the freeto bound ratio of cortisol is highly variable and that during the acute phase response direct quantification of free bioactive cortisol concentrations seems to be biologically more appropriate than the measurement of total cortisol concentrations.