The role of Cu2+ in nicotine nitrosation reaction was investigated using High-Performance Liquid Chromatography (HPLC), Nuclear Magnetic Resonance (NMR) and UV-vis spectroscopy. The nitrosation reaction of nicotine was carried out in both “clean” citrate phosphate buffer and in tobacco matrix. Titration with Cu2+ monitored by NMR and pH dependent UV-vis profiles strongly indicate that Cu2+ binds irreversibly to the pyridine nitrogen of nicotine. Studies on nitrosation kinetics reveal that in presence of Cu2+, the activation energy of the nitrosation reaction was increased suggesting unfavorable nitrosamine-formation kinetics. NMR and kinetics data strongly suggest that by binding irreversibly with the pyridine moiety of nicotine, Cu2+ imparts partial inhibition of nitrosamine formation from nicotine. This presentation will demonstrate the extent of nicotine nitrosation and role of copper on in the nitrosation reaction.