Several N-nitroso compounds, present in foods and beverages or formed in the stomach from their precursors, act as alkylating agents. By using a highly reliable technique (high-resolution gas chromatography-mass spectrometry with negative-ion chemical ionization and selected ion recording), we measured a series of specific O6-alkylguanines in snap-frozen paired stomach tissue samples (tumor and noninvolved mucosa) obtained at surgery from 24 gastric cancer patients identified in Florence, Italy. Samples of noninvolved mucosa had higher levels of total O6-alkylguanines and more frequently detectable levels (54%) than tumor samples (29.2%). O6-propylguanine and O6-methylguanine were the single adducts most frequently detected in noninvolved mucosa and tumor tissue, respectively. Tumor samples showed higher levels of total O6-alkylguanines in female patients (p = 0.03) and among those with a diffuse histological type (p = 0.06) or seronegative for Helicobacter pylori CagA antibodies (p = 0.06). Mean dietary nitrate intake was significantly higher in patients with detectable levels of adducts in tumor samples (p = 0.03). Estimated intakes of dimethylamine and N-nitrosodimethylamine correlated with total levels of O6-alkylguanines in noninvolved gastric mucosa. These findings, although based on a small series of cases, support a role for N-nitroso compounds from dietary sources in the etiology of gastric cancer.