We report here that tobacco-specific nitrosamines (TSNAs) are produced from specific tobacco alkaloids during water chloramination. To identify the specific precursors for the formation of specific TSNAs in drinking water, we have developed a solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method for simultaneous determination of five TSNAs and three tobacco alkaloids. Using this method, we detected nicotine (NIC) at 15.1 ng/L in a source water. Chloramination of this source water resulted in the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (0.05 ng/L) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) (0.2 ng/L) along with the reduction of NIC to 1.1 ng/L, suggesting that NNK and NNAL were formed from NIC. To confirm that tobacco alkaloids are the precursors of TSNAs, we chloraminated water-leaching samples of tobacco from three brands of cigarettes and found that the formation of TSNAs coincides with the reduction of the alkaloids. Chloramination of individual alkaloids confirms that NNK and NNAL are produced from NIC, N-nitrosonornicotine (NNN) from nornicotine (NOR), and N-nitrosoanabasine (NAB) from anabasine (ANA). Furthermore, we have identified specific intermediates of these reactions and proposed potential pathways of formation of TSNAs from specific alkaloids. These results confirm that NNK and NNAL are the disinfection byproducts (DBPs) resulting from NIC in raw water.