Metabolism of four tobacco-specific N-nitrosamines (TSNAs), N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has been studied by solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). 4-(Methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) was used as internal standard. SPE and LC-MS-MS was found to be a rapid, simple, sensitive, and selective method for analysis of TSNAs in rabbit serum. The relative standard deviation (R.S.D., n = 6) for analysis of 5 ng mL(-1) and 0.5 ng mL(-1) standards and of serum sample spiked with 5 ng mL(-1) standards of five TSNAs was 2.1-11% and recovery of 5 ng mL(-1) standards from serum was 100.2-112.9%. A good linear relationship was obtained between peak area ratio and concentration in the range of 0.2-100 ng mL(-1) for NNAL and 0.5-100 ng mL(-1) for other four TSNAs, with correlation coefficients (R2) >0.99 (both linear and log-log regression). Detection limits for standards in solvent were between 0.04 and 0.10 ng mL(-1). Doses of TSNAs administered to rabbits via the auricular vein were 4.67 microg kg(-1) and 11.67 microg kg(-1), in accordance with the different levels in cigarettes. Metabolic curves were obtained for the four TSNAs and for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of NNK; on the basis of these curves we modeled metabolic kinetic equations for these TSNAs by nonlinear curve fitting.