Background: Linezolid is the first synthetic compound of a new group of antimicrobials, the oxazolidinones, which inhibit protein synthesis. It shows a broad spectrum of activity against Gram positive organisms. With respect to its pharmacokinetics, linezolid shows a relatively high volume of distribution and good penetration into inflammatory fluids, bone, fat and muscle. Methods: A reversed-phase isocratic high-performance liquid chromatographic method for linezolid analysis in piglet pulmonary tissue is described. Tissue samples and controls were prepared in 1×TBE (1 M Tris, 0.9 M boric acid, 0.01 M EDTA). The mobile phase consisted of 20% ultrafiltered water and 80% of (A) 15 mM potassium monohydrogen phosphate buffer (pH=5) with (B) acetonitrile (80%/20%; v/v). Samples were homogenized and precipitated with HClO4 3% (1/1, v/v). The injection volume was 100 μL. Ofloxacin was used as an internal standard. Results: The assay was linear over a linezolid concentration range: 1.6-100 μg/mL. The method provided good validation data (n=15): inaccuracy (3.6%), intra and inter-day variability (4.2% and 5.2%, respectively), recovery (91.8%), limit of detection (0.8 μg/mL) and quantitation (1.6 μg/mL) and acceptable stability within 24 h in the auto-sampler. Conclusions: The method offers a fast and simple approach to determine linezolid in pulmonary tissue which could be of use in pharmacokinetic studies. © 2010 by Walter de Gruyter.