Tobacco-specific nitrosamines (TSNAs) are carcinogens that accumulate in tobacco leaves during curing, storage, and processing, and their amounts in processed tobacco vary dependent on several intrinsic and extrinsic factors. Here, we assessed the hypothesis that there is a link between reactive oxygen species levels in leaves and TSNA formation during curing. First, we show that burley varieties KT 204LC and NCBH 129LC accumulate TSNAs to different levels but not as a result of a variety-specific abundance of TSNA precursors. Next, we measured the levels of reactive oxygen species, and we show that the variety that accumulates more TSNAs, NCBH 129LC, had significantly higher levels of hydrogen peroxide than KT 204LC. The NCBH 129LC also has more oxidatively damaged and glutathionylated proteins. Finally, we analyzed the antioxidant levels in KT 204LC and NCBH 129LC and their tolerance to oxidative stress. NCBH 129LC contained more of the essential antioxidant glutathione and was more tolerant to the oxidative stress-generating compound paraquat. Collectively, our data suggest that there is indeed a link between foliar oxidative stress parameters and the extent to which TSNAs accumulate in cured tobacco leaves.Copyright © 2021 Elsevier GmbH. All rights reserved.