This work was performed to develop an experimental animal model for the study of antibiotic drug distribution into middle ear ¯uid (MEF) and to evaluate its relevance and signi®cance to the clinical treatment of otitis media (OM). Chinchillas were assigned to normal or infected ear groups after Eustachian tube obstruction (ETO) or direct trans-bullar inoculation with type 3 Streptococcus pneumoniae. Following survival surgery to implant microdialysis (MD) probes in the jugular vein and middle ear (ME), amoxicillin was given intravenously (iv) as a bolus or infusion. Drug concentrations in blood and MEF were continuously monitored by microdialysis. The measured concentrations were corrected for probe recovery by simultaneous retrodialysis. Multiple MEF and blood sampling was also performed to validate the animal model and MD sampling technique. Bacterial infection was successfully induced 3±7 days after the inoculation, whereas the control group gave negative bacterial culture results. The b-lactam antibiotic, amoxicillin, exhibited an elimination half-life of 0.33AE0.23 h (n 9) in chinchilla blood, 1.46AE0.50 h (n 5) and 1.75AE0.84 h (n 4) in MEF of normal and infected ears (p 0.6), respectively. MEF-to-blood amoxicillin concentration ratios at steady state following iv infusion were 0.26AE0.06 (n 5) and 0.28AE0.11 (n 4) for normal and infected ears (p 0.7), respectively. MD allows continuous monitoring of drug concentration±time pro®les in blood and MEF in an awake chinchilla model. The concentrations measured by MD were validated by direct sampling. The ratio of the area under the curve (AUC) of drug concentration in MEF versus time to that in blood after iv bolus doses was less than unity, as was the steadystate concentration ratio following constant-rate iv infusion, suggesting an active transport mechanism was involved in the ef¯ux of amoxicillin from the ME of chinchilla. The results of studies involving infected ears were not signi®cantly different from those in normal ears in terms of amoxicillin distribution across the ME mucosal membrane after systemic administration.