The aim of the present study was to investigate amoxicillin (AMX) distribution in muscle interstitial fluid by microdialysis in healthy, awake rats. Microdialysis probes were inserted into the jugular vein and hind leg muscle. Probe recoveries in each rat were determined by retrodialysis with cefadroxil. AMX was administered as a bolus dose of 50 mg.kg(-1), and microdialysis samples were collected during 180 min. Concentrations of unbound drug in blood and muscle were analyzed simultaneously by a population approach. Simulations were conducted using a hybrid, physiologically based pharmacokinetic model to investigate the potential impact of tissue blood flow on muscle AMX distribution. A two-compartment pharmacokinetic model described adequately the unbound amoxicillin concentration-time profiles in blood and muscle. Muscle AMX distribution equilibrium was rapidly achieved. Consequently, the best results were obtained by considering concentrations in muscle as part of the central compartment. The ratio of the concentration of unbound drug in muscle to that in blood (Rmodel) was estimated to 0.80 by the model, which is close to the mean value obtained by noncompartmental data analysis (Rarea= 0.86 +/- 0.29). Simulations conducted with a hybrid, physiologically based pharmacokinetic model suggest that a muscle blood flow reduction of 30% to 50%, such as could be encountered in critical care patients, has virtually no effect on muscle AMX concentration profiles. In conclusion, this study has clearly demonstrated that AMX distributes rapidly and extensively within muscle interstitial fluid, consistent with theory, and that altered muscle blood flow seems unlikely to have a major effect on these distribution characteristics.