This study was conducted to determine the amount of unprotonated nicotine in cigarette mainstream smoke and to investigate its relationship to level of filter ventilation and machine smoking conditions. Unprotonated nicotine from TPM trapped on a Cambridge filter pad(CFP) was adsorbed by Carboxen/PDMS SPME fiber, thermally desorbed and determined by GC/MS. 2R4F reference cigarette, twelve commercial brands from the Korean market and five test cigarette samples, which had the same tobacco blend with different levels of filter ventilation, were analyzed for unprotonated nicotine. In commercial brands, the amount of unprotonated nicotine changed slightly depending on the pH values of smoke, and decreased as the tar level increased. filter ventilation in these commercial cigarettes was $28{sim}80%$ and the higher filter ventilation increased relative unprotonated nicotine levels, but not significantly. However, in five test cigarettes with different filter ventilation$(0{sim}70%)$, unprotonated nicotine levels increased almost linearly with the level of filter ventilation. Concentrations of unprotonated nicotine in mainstream smoke generally increased in order $HC;<;ISO;{leq};MDPH$ machine smoking conditions. The ratio of unprotonated nicotine to total nicotine among $cigarettes({alpha}_{fb})$ increased in order RC < MDPB < ISO conditions. Concentrations of unprotonated nicotine varied with three machine smoking conditions.