Substantial quantities of the carcinogenic tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (1; NNK) are still found in the mainstream smoke of tobacco exhaustively extracted with water, indicating the presence of an insoluble, matrix-bound form. Soluble and matrix-bound concentrations of 1 in tobacco were determined by applying a new method using sequential aqueous extraction at room temperature and at 130 °C. On average, 77% and 53% of the total content of 1 were matrix-bound in air-cured (Burley type) and flue-cured tobaccos, respectively. Thermal release of 1 from its matrix-bound form above ca. 200 °C can account for a large fraction of its concentration in cigarette mainstream smoke. An already matrix-bound alkaloid precursor of matrix-bound 1 was identified in vascular tissue of green leaf midribs. The incubation of vascular cell-wall preparations with the lignin precursor coniferyl alcohol and isotopically labeled nicotine or pseudooxynicotine (2) led to the formation of labeled matrix-bound 1 after nitrosation, suggesting that incorporation of nicotine or its oxidized product 2 during lignin polymerization is the origin of the formation of matrix-bound 1.