The objective of this study was to validate subcutaneous (sc) microdialysis sampling to study¯urbiprofen pharmacokinetics and plasma protein binding in the awake freely moving rat. A linear microdialysis probe was manufactured using a Hemophane 1 hollow ®ber which was tested in vitro and in vivo for the recovery of urbiprofen and naproxen used as retrodialysis marker. Flurbiprofen was administered intraperitoneally and intravenously at a dose of 20 mg/kg in rats. In both cases, conventional blood sampling and sc microdialysis sampling were simultaneously performed. The microdialysates were analyzed on-line by high-pressure liquid chromatography. Naproxen, which was shown to have a similar in vivo loss by retrodialysis as¯urbiprofen (71.5 AE 0.9% and 71.0 AE 0.8% respectively, n 3), was used to continuously monitor probe recovery. Concentration-dependent protein binding of urbiprofen was demonstrated in vivo based on experiments with a simultaneous sc microdialysis and blood sampling. Values of unbound fraction were similar to those reported previously by intravenous microdialysis sampling, demonstrating that the sc unbound concentrations are very similar to those in the central compartment. There was no signi®cant difference among pharmacokinetic parameters (AUC, CL, t 1/2z , Vd) for total or unbound¯urbiprofen determined after intraperitoneal and intravenous administration. Subcutaneous microdialysis is a simple yet powerful tool to study the pharmacokinetics and the in vivo plasma protein binding of¯urbiprofen in the awake unrestrained rat. ß