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To determine the concentration–time curves of linezolid in serum and vitreous from 24 patients undergoing vitrectomy.Vitrectomy was performed 1, 2, 4, 8 and 12 h after infusion of 600 mg of linezolid in 20 patients divided into groups of four. Four additional patients were studied 12 h after two separate oral doses of 600 mg of linezolid. Serum samples were obtained 1 h after linezolid administration to determine Cmax; vitreous and a second serum sample were taken simultaneously during the vitrectomy in all patients, and the concentrations of linezolid in vitreous (Cv) and serum (Cs) were determined.Among patients who received one intravenous dose of 600 mg of linezolid, the highest mean Cv was observed at 4 and 8 h following linezolid administration (3.4 and 3.7 mg/L). The highest mean Cv was observed in patients who received two oral doses of 600 mg of linezolid separated by 12 h (4.5 mg/L), which was higher than the MIC90 for Staphylococcus epidermidis. The highest Cv/Cs ratio was reached 12 h after administration of one and two doses (2.4 and 1.5, respectively).Microbiologically significant concentrations of linezolid can be achieved in the vitreous of the non-inflamed human eye after intravenous administration of 600 mg, and it is even better after two doses of 600 mg. It appears that linezolid accumulates in the vitreous, achieving potentially useful steady-state concentrations. An evaluation of clinical efficacy is needed to confirm the perceived utility based on the pharmacokinetics.