A high-performance liquid chromatographic method for the determination of vancomycin in rabbit serum, vitreous and aqueous humour has been developed. No clean-up step was necessary for vitreous and aqueous humour samples. For serum samples liquid-liquid and solid-phase extraction were tested and the best results were achieved using C18 cartridges. The extracts were analyzed on a C18 reversed-phase column, using a mixture of 0.05 M phosphate buffer (pH 4) with 10% of acetonitrile as mobile phase. The detection was carried out at 198 nm, which allows higher sensitivity. The average quantitation limit obtained was 0.03 micrograms/ml. The method has been applied to the study of the residual quantities of vancomycin in serum and rabbit eyes after intravitreal administration of the drug in endophthalmitis treatment.