Tobacco-specific nitrosamine (TSNA) accumulation during burley curing is probably driven by endophytic microbial anaerobic denitrification. This study investigates the effect of time of spraying a soil suspension onto greenhouse-grown plants that otherwise do not accumulate TSNAs. A high converter selection of the variety TN 90 was grown in soilless media in a greenhouse far removed from any commercial tobacco production facilities. Plants were moved to a second greenhouse and sprayed at topping, at 14 days after topping, and four hours before harvest, with a filtered suspension of soil collected from a tobacco field. Half of the plants of each spray treatment were cured in a commercial tobacco barn and half in a curing structure in the greenhouse. Half of the lamina from each cured leaf was air-dried for TSNA and alkaloid analysis and the other half freeze-dried for microbial analysis. These greenhouse-grown plants did not develop to the size of field-grown plants and took 15 weeks to fully cure. The total TSNAs were low in all treatments, ranging from 0.4 to 1.2 ppm. Total TSNAs in the greenhouse-cured plants were greater than in the barn-cured plants, and declined as the interval between the soil suspension application and harvest shortened, but not in the barn-cured plants. Nitrosoanatabine (NAT) declined as the interval between the soil suspension application and harvest shortened at both sites, Nitrosonornicotine (NNN), however, was significantly higher in the greenhouse-cured plants than those cured in the barn, but there were no differences between times of application. These differences could be attributed to the difference in the curing conditions: the temperature in the greenhouse was consistently higher than in the barn, but the humidity was lower. (Reprinted with permission)