The aim of this study was to develop a specific and sensitive high-performance liquid chromatographic assay for the determination of linezolid in human plasma, and bronchoalveolar lavage. The sample extraction was based on a fully automated solid-phase extraction with an OASIS HLB cartridge. The method used ultraviolet detection set at a wavelength of 254 nm and a separation with a Zorbax Eclipse XDB C8 column. The assay has been found linear over the concentration range 0.02-30 microg/ml and 0.04-30 microg/ml for linezolid, respectively, in plasma and bronchoalveolar lavage. It provided good validation data for accuracy and precision (CV <4.64% and 5.08%, accuracy in the range 96.93-102.67% and 97.33-105.67%, respectively, for intra- and inter-day). The assay will be applied to determine the penetration of linezolid in human bronchoalveolar lavage during pharmacokinetic steady-state.