Linezolid is a new, promising antibacterial agent to treat severe infections. A rapid HPLC assay using UV detection for the determination in microdialysate and human plasma was developed. After sample preparation, using acetonitrile for plasma and water for microdialysate, 20 microl was injected and separated on a RP-18 column. Overall, the assay exhibited good precision and accuracy. The diffusion properties of linezolid investigated in in vitro microdialysis experiments revealed a mean relative recovery of 77.5% (CV: 5.4%; delivery and recovery experiments). Following characterization of linezolid in in vitro microdialysis, the setting is suitable for application in clinical studies.