The plasma protein binding and competitive inhibition parameters of R(-)- and S(+)-ibuprofen were determined in vivo in 12 healthy subjects. Subjects participated in a 4 x 4 Latin square design in which oral solutions of drug were administered as 300 mg R(-)-ibuprofen, 300 mg S(+)-ibuprofen, 300 mg R(-)- + 300 mg S(+)-ibuprofen, and 300 mg R(-)- + 600 mg S(+)-ibuprofen. Unlabeled ibuprofen enantiomers were quantitated using a stereospecific reversed-phase HPLC assay, and plasma protein binding experiments were performed using radiolabeled 14C-enantiomers and an ultrafiltration method at 37C. At therapeutic drug concentrations, the protein binding of each enantiomer was greater than 99%. Furthermore, the binding of ibuprofen enantiomers was stereoselective and mutually competitive, as well as nonlinear. The bound-free data were fitted to a model in which the non-linearity of plasma protein binding and competition between enantiomers for binding sites could be accommodated. There were substantial differences in the affinity of ibuprofen enantiomers for protein binding sites (RP2 = 0.358 +/- 0.185 vs. SP2 = 0.979 +/- 0.501 micrograms/ml; mean +/- SD) but no differences in their binding capacity (RP1 = 160 +/- 86 vs. SP1 = 161 +/- 63 micrograms/ml). Although statistically significant, the differences in competitive inhibition parameters were more modest (SKI = 0.661 +/- 0.363 vs. RKI = 0.436 +/- 0.210 micrograms/ml). As a result, the intrinsic binding (i.e., P1/P2) of R(-)-ibuprofen was greater than S(+)-ibuprofen, and the unbound fraction was significantly greater for S-enantiomer vs. R-enantiomer after a given dose of R-ibuprofen or racemate.