Down-regulating nicotine demethylase (ND) activity in tobacco using RNAi interference mechanisms or by mutating the ND gene reduces nornicotine formation and should lead to lowering N-nitroso-nornicotine (NNN) levels in cured leaf. Over the past two years, we have conducted field trials at multiple locations to verify and quantify NNN and tobacco specific nitrosamine (TSNA) reduction in cured leaf obtained from Burley and dark tobacco genotypes that were engineered or mutated for reduced ND activity. RNAi transgenic lines of TN 90 Burley tobacco and NL Madole dark tobacco were grown in replicated trials at several sites in Kentucky in 2007 and 2008. Also included in the trials were the control varieties TN 90LC, NL Madole LC, the high converter versions of Burley and dark tobaccos, and two lines of TN 90 that possess a mutated, non-functional version of ND. The mutated lines were in the M2 (2007) or M2BC1 (2008) generation and were selected from a population treated with the mutagen EMS. There were no significant differences in leaf size or number, plant height, maturity or yield between any of the transgenic lines and their respective controls. The reduced morphological traits and leaf yield of the mutated lines were likely the result of an insufficient number of backcrosses needed to eliminate the random mutations caused by the EMS treatment. The reduced cured leaf yield of the mutated lines suggests more backcrossing is needed to eliminate probable random mutations caused by the EMS treatment. Nicotine conversion rates were significantly less in the transgenic and the mutated lines than in the respective LC lines. The levels of NNN measured in the cured leaf of the TN 90 transgenic lines were reduced by two to five times of those found in the controls. Leaf NNN measured in the cured leaf of the mutant lines had NNN levels that were also less than the control. The transgenic and mutation approaches to down-regulating ND activity are effective in reducing a major component of TSNA's in tobacco.