A three-year study from 2012-2014 was done to test the effects of temperature and relative humidity (RH) on TSNA (tobacco specific nitrosamine) accumulation in air-cured Burley tobacco. Twenty-four curing chambers were constructed, each holding six sticks of six stalk-cut plants. The temperature and humidity in each unit could be controlled independently of each other at constant levels for the duration of the cure and were monitored with data loggers. The tobacco was cured in the units at all combinations of three temperatures (16, 24 and 30 °C) and three humidities (60, 75 and 90% in 2012, and 50, 65 and 80% in 2013 and 2014). Each year, an additional treatment was cured in a conventional air-cured barn. In 2012, the variety was TN 90H (high converter selection of TN 90); in 2013, TN 90H and TN 90LC (commercial low converter); and KT 210 in 2014. Samples for leaf chemistry analysis were taken after the end of the cure each year, and also after 14 and 35 days in the 2014 test. Data were analyzed as a multiple regression of leaf chemistry parameters against RH and temperature. Conversion of nicotine to nornicotine was not affected by either temperature or RH in any of the three years. The multiple regression for N’-nitrosonornicotine (NNN) was significant in all years and all varieties. The RH component of the multiple regression was highly significant in all cases, but the temperature component was only significant in KT 210 in 2014, although it was almost significant in both varieties in 2013.