Blocking or down-regulating the conversion of nicotine to nornicotine is a major strategy for preventing the formation of N'-nitrosonornicotine (NNN), a tobacco specific nitrosamine formed by microbial action during tobacco curing and processing. Previously, we described the cloning and characterization of the gene responsible for nicotine demethylation (Nicotine Demethylase). Here, we report on two strategies used to down-regulate this gene. First, we created GMO tobacco containing RNAi constructs, and secondly, we identified lines with mutated versions of nicotine demethylase from a converter population that had been treated with a mutagen (EMS). The converter levels in some of the transgenic lines containing RNAi constructs were as low as 0.1% under greenhouse or field conditions. This low conversion level remained stable up through three generations of self-pollination. In addition, nineteen lines were identified having EMS-induced mutations in the nicotine demethylase gene. Four of these lines had a knock-down phenotype (0.2-1.3% conversion rates) with one line having a nonsense mutation at amino acid position 329 and another line having a missense mutation at amino acid position 107. Experiments to detect nicotine conversion rates in the M1, M2 and M3 generation of the EMS mutant plants showed they were stable, and the conversion rates remained low. We also crossed two of these mutant lines with seven commercial varieties and two converter counterparts of dark and Burley tobaccos. Results from progeny tests provided a genetic pattern of single gene mutation. Sequencing data determined that the Nicotine Demethylase gene had been permanently mutated. RT-PCR assays on the lines containing the truncated gene showed that mRNA expression was decreased or even abolished, while mRNA in missense mutant lines was indeed changed to a mutated version expressed as highly as in the wild-type converter lines.