Nicotine is the most abundant alkaloid in cultivated tobacco (Nicotiana tabacum), typically constituting more than 90% of total alkaloids. The remaining fraction consists of three minor alkaloids: nornicotine, anabasine and anatabine. Genetic control of nicotine biosynthesis in tobacco and subsequent development of low alkaloid traits have been derived historically from nic1nic2 mutant lines. This unique genetic resource has been used to successfully produce tobacco lines having approximately 95% nicotine reduction compared to a Nic1Nic2 control. However, the quality of cured leaf from these mutant lines, as measured by the USDA Grade Index, is commercially undesirable. The objective of this study was to develop new ultra-low nicotine tobacco lines having nicotine levels comparable to nic1nic2 mutants while maintaining a USDA Grade Index of cured leaf comparable to a Nic1Nic2 control. PR50, a key nicotine biosynthetic pathway regulator, and Putrescine N-methyltransferase (PMT), the enzyme involved in the first committed step of nicotine biosynthesis, were used. Transgenic tobacco plants containing separate RNAi constructs of PR50 and PMT were generated and grown in the field. After harvest and curing, alkaloid levels and cured leaf Grade Indexes were measured. Cured leaf nicotine levels in PMT-RNAi lines and PR50-RNAi lines were reduced approximately 95% and 80-90%, respectively, compared to Nic1Nic2 controls. In addition, cured leaf samples of PMT-RNAi and PR50-RNAi lines had quality Grade Indexes similar to Nic1Nic2 controls and significantly higher than samples from nic1nic2 mutant lines.