Our goal is to develop tobacco lines that display a greatly reduced propensity for producing tobacco-specific nitrosamines (TSNAs) during the curing, storage and processing of the leaf. Specifically, we are targeting the genes responsible for the demethylation of nicotine as a strategy for minimizing the synthesis and accumulation of nornicotine, the alkaloid precursor of N-nitrosonornicotine (NNN), a TSNA classified as a Group I carcinogen by the International Agency for Research on Cancer. Several years ego we identified CYP82E4 as the genetic locus responsible for the exceptionally high levels of nicotine demethylation observed in tobacco plants possessing an activated converter locus (termed "converters"). We subsequently discovered two "minor" nicotine demethylase genes, CYP82E5v2 and CYP82E10 , the combined activity of which is responsible for the majority of the low level of nornicotine observed in a typical nonconverter tobacco plant (2 - 4% of total alkaloid content). By combining debilitating EMS-induced mutations for all three nicotine demethylase loci into single genotypes, we have been able to develop lines that accumulate exceptionally low levels of nornicotine. These materiels promise to provide a facile and effective means of producing tobacco products with greatly reduced NNN content. In this presentation we will provide an update of our latest research results.