ACE is a popular technique for evaluating association constants between drugs and proteins. However, ACE has not previously been applied to study the association between electrically neutral biomolecules and plasma proteins. We studied the affinity between human and bovine serum albumins (HSA and BSA, respectively) and three neutral endogenous steroid hormones (testosterone, epitestosterone and androstenedione) and two synthetic analogues (methyltestosterone and fluoxymesterone) by applying the partial-filling technique in ACE (PF-ACE). From the endocrinological point of view, the distribution of endogenous steroids among plasma components is of great interest. Strong interactions with albumins suppress the biological activity of steroids. Notable differences in the association constants were observed. In the case of the endogenous steroids, the interactions between testosterone and the albumins were strongest, and those between androstenedione and the albumins were substantially weaker. The association constants, K-b, for testosterone, epitestosterone and androstenedione and HSA at 37 degrees C were 32 100 +/- 3600, 21600 +/- 1500 and 13 300 +/- 1300 M-1, respectively, while the corresponding values for the steroids and BSA were 18 800 +/- 1500, 14000 +/- 400 and 7800 +/- 900 M-1. Methyltestosterone was bound even more strongly than testosterone, while fluoxymesterone was only weakly bound by the albumins. Finally, the steroids were separated by PF-ACE with HSA and BSA used as resolving components.