Racemic licarbazepine (Lic) is the active metabolite of oxcarbazepine (OXC) and eslicarbazepine acetate (ESL), appearing in human plasma as S-licarbazepine (S-Lic) and R-licarbazepine (R-Lic). However, human metabolism of OXC and ESL to Lic differs in the S-Lic/R-Lic enantiomeric ratio observed in plasma. S-Lic appears in higher proportion after ESL administration than after OXC (95% versus 80%). Enantioselective pharmacokinetics of Lic enantiomers have been found in mice after their separate administration and in humans following OXC treatment. Since protein binding of drugs may be enantioselective and a determining factor of pharmacokinetics, the binding of S-Lic and R-Lic to mouse and human total plasma proteins and, specifically, to human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP) were herein investigated for the first time. Free and bound fractions of S-Lic and R-Lic were separated by ultrafiltration after previous in vitro incubation of spiked plasma samples and protein solutions with each enantiomer at 10, 25 and 50 mu g/ml. The results revealed that the extent of binding of Lic enantiomers to total plasma proteins was 30% and independent of the drug concentration and species considered. The data also suggest that the binding of Lic enantiomers to HSA is greater than that to AGP. Moreover, absence of enantioselectivity in the binding of Lic enantiomers to mouse and human plasma proteins and to HSA and AGP is evident. In conclusion, these findings suggest that the enantioselectivity observed in vivo in the biodisposition of S-Lic and R-Lic is not dependent on their affinity to plasma proteins. Copyright (C) 2010 John Wiley & Sons, Ltd.