Regulations under consideration by the U.S. Food and Drug Administration and the World Health Organization propose that nicotine concentration in tobacco (Nicotiana tabacum L.) should be lowered to non-addictive levels (0.3 to 0.5 mg g −1 ). The proposed standards are 90 to 95% lower than the nicotine concentration typically documented in commercially available cultivars. Research was conducted in six environments to evaluate two cultivars with normal alkaloid levels (K326 and NC95) and four genotypes with low alkaloid levels (DH16A, DH22A, DH32, and LAFC53). Each cultivar and genotype was paired with three N application rates: 70, 85, and 100% of the recommended rate. As N application declined, so too did cured leaf yield and nicotine, anabasine, and anatabine concentration in K326 and NC95. These factors were generally not affected by N application in the low alkaloid genotypes. In contrast, LAFC53 consistently produced the lowest cured leaf quality, value, and reducing sugar concentration when compared to all other cultivars. This observation demonstrates that K326 isolines are agronomically superior to LAFC53. Despite reductions in nicotine, the lowest documented concentration was still 10fold greater than the proposed minimum (LAFC53). Nitrogen did not influence the measured parameters as much as genetics; therefore, additional research that involves other agronomic practices is warranted. In addition, further genetic manipulation will be required to meet the standards proposed by regulatory groups.