Research conducted during past decades to reduce the level of the tobacco specific nitrosamine N-nitrosonornicotine (NNN) and its precursor nornicotine in tobacco yielded identification of three tobacco genes encoding for cytochrome P450 nicotine demethylases converting nicotine to nornicotine. We carried out trials to investigate the effect of using tobaccos containing three non-functional nicotine demethylase genes on the selective reduction of NNN in cigarette tobacco filler and mainstream smoke. Our results indicate that the presence of non-functional alleles of the three genes reduces the level of nornicotine and NNN in Burley tobacco by 70% compared to the level observed in currently available low converter (LC) Burley tobacco varieties. The new technology, named ZYVERT™, does not require a regular screening process, while a yearly selection process is needed to produce LC Burley tobacco seeds for NNN reduction. The reduction of NNN observed in smoke of blended prototype cigarettes is proportional to the inclusion level of tobacco having ZYVERT™ technology. Inclusion of Burley tobacco possessing the new trait into a typical American blend resulted in a selective reduction of NNN in cigarette smoke, while the levels of other Harmful and Potentially Harmful Constituents (HPHC) currently in the abbreviated list provided by the US Food and Drug Administration are statistically equivalent in comparison with the levels obtained in reference prototype cigarettes containing LC Burley.