Objectives: The oxazolidinone class of antibiotics such as linezolid have a narrow spectrum of activity that targets Gram-positive bacteria. We hypothesized that the poor activity of linezolid in Gram-negative bacteria is in part caused by relatively low intracellular concentration due to efflux. Methods: Using whole cell accumulation assays we estimated the intracellular concentration of linezolid in Escherichia coli and other Enterobacteriaceae. We included test strains with enhanced RND-type multidrug efflux pump activity and with genetic inactivation of the pump or functional inhibition by carbonyl cyanide m-chlorophenylhydrazone as inhibitor of the proton motive force or 1-(1-naphthylmethyl)-piperazine (NMP), an efflux pump inhibitor. Results: Consistent with susceptibility studies, enhanced pump activity caused decreased accumulation, and pump inactivation and inhibition caused increased accumulation, of linezolid. The accumulation levels in test strains of E. coli, Citrobacter freundii and Enterobacter aerogenes with functional pumps were lower than in control strains of Staphylococcus aureus and Enterococcus faecium, but were higher after pump inactivation and correlated with ethidium bromide and pyronin Y accumulation. Conclusions: The intracellular concentration of linezolidis comparatively low owing to efficient efflux of the drug and could be increased substantially by inhibition of RND-type efflux pumps. © The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.