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Objective To investigate the pharmacokinetics of cycloserine in healthy rat blood and lung tissues. Method Healthy rat blood and lung tissues were sampled in vivo by microdialysis sampling technique simultaneously. The concentrations of cycloserine in both blood and lung tissues were measured by high performance liquid chromatography mass spectrometry. All data were analyzed by Win Nonlin software. Results:The maximum concentration of free cycloserine in blood and lung tissue were (10.61 ± 2.42) mg/L and (1.53 ± 1.71) mg/L at 1h. And then both continued to decline. After administration the concentrations of free cycloserine in the blood has been higher than the concentrations in lung tissues. The area under the concentration curve (AUC) of free cycloserine was (33.53 ± 6.51) h·mg-1·L-1 in blood, and (4.49 ± 2.08) h·mg-1·L-1 in lung tissues. Conclusion Microdialysis sampling technique combined with high performance liquid chromatography mass spectrometry / mass spectrometry can be accurately and objectively reflect the drug in the blood and tissues of the pharmacokinetic characteristics of cycloserine. Concentrations in lung tissues were significantly lower than concentrations in blood.