BACKGROUND It was reported that tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was a powerful pulmonary carcinogen, predominantly inducing adenocarcinoma of the lung in mouse. The aim of this study is to assay metabolites of NNK, which are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its O-glucuronide (NNAL-Gluc), and their ratio (NNAL-Gluc/NNAL) in smokers and non-smokers' urine, and to explore the carcinogenicity of NNK among different people. METHODS Using high pressure liquid chromatograph (HPLC) and gas chromatograph-mass tadom (GC-MS/MS), NNAL-Gluc and NNAL in 24h urine were detected in 8 healthy smokers, 10 lung cancer smokers and 4 healthy non-smokers. RESULTS Both of the two metabolites were not found in non-smokers' urine. The ratios of urine NNAL-Gluc/NNAL were greatly different among different smokers. The mean ratio of NNAL-Gluc/NNAL in healthy smokers was 4.95, and 0.5 in lung cancer smokers. CONCLUSIONS The results provide the first evidence for metabolite detection of tobacco-specific nitrosamine in Chinese smokers' urine . The result suggests that detoxification ability of healthy smokers is higher than that of lung cancer smokers. It may provide a detective way to screen high risk people for lung cancer in smokers.