The binding of the enantiomers of indobufen (INDB) to human serum proteins was investigated using the racemic mixture or the pure (+)-S-enantiomer in a concentration range of 2.5–100.0 mg/L. In addition, the pharmacokinetics of free (unbound) and total INDB enantiomers were studied 1) following administration of a single 200 mg rac-INDB tablet to healthy volunteers, and 2) in obliterative atherosclerosis patients at steady state. The free fraction of INDB was obtained by ultrafiltration. Using the racemic mixture, the binding parameters of the two enantiomers were different, showing enantioselectivity in protein binding. The (−)-R-enantiomer was bound more strongly to human serum albumin, with association constant K = 11.95 ± 0.98 × 105 M−1 and n = 0.72 ± 0.02 binding sites. The comparable data for the (+)-S-enantiomer were K = 4.65 ± 0.02 × 105 M−1, n = 0.92 ± 0.01. When the binding of (+)-S-enantiomer was studied alone, the association constant K (2.10 ± 0.18 × 105 M−1) was lower and the number of binding sites was increased, to n = 1.87 ± 0.17. Competition occurred between the enantiomers, with the (−)-R-enantiomer displacing its antipode. The fraction of both enantiomers bound to serum proteins was 99.0%, which increased with decreasing initial concentration of the enantiomers. In healthy volunteers the (+)-S-enantiomer was eliminated faster than its (−)-R antipode, resulting in a lower AUC for the (+)-S-enantiomer. Significant differences were observed in the total INDB enantiomer concentrations. The mean unbound fraction of (−)-R- and (+)-S-INDB was 0.45% and 0.43%, respectively. Levels of the free (+)-S-enantiomer were higher than its (−)-R-antipode at steady state in patients with obliterative atherosclerosis who also took other drugs. The free enantiomer fraction increased to around 1% upon repeated administration. We conclude that the more rapid elimination of the (+)-S enantiomer is associated with its weaker binding to serum proteins. Chirality 14:736–741, 2002. © 2002 Wiley-Liss, Inc.