Aims: To examine the hypothesis of non-immune defence mechanisms based on nitrite. Methods and Results: The acidified media (nutrient broth or citrate-phosphate buffer) under aerobic conditions with additions of physiological levels of nitrite, L-ascorbic acid, iodide and thiocyanate were used to simulate gastric juice. The bactericidal effects of acidified nitrite on Escherichia coli and lactobacilli were investigated using bacterial plate counts. Conversion of acidified nitrite to nitric oxide, nitrogen dioxide and nitrate was also studied. Nitrite significantly increased the bactericidal effects on E. coli and lactobacilli. The bactericidal effects were enhanced by thiocyanate but not by L-ascorbic acid and iodide. L-Ascorbic acid and thiocyanate, but not iodide, enhanced the decomposition of acidified nitrite in nutrient broth. Acidified nitrite was converted to both nitric oxide and nitrate, but a portion of the acidified nitrite in nutrient broth may have been converted to other unidentified nitrogen compounds. Nitrogen dioxide was not detected in any of the samples. Conclusions: The bactericidal effects of nitrite appeared to be primarily related to nitrous acid, and possibly to other unidentified nitrogenous metabolites, but not to nitric oxide and nitrogen dioxide. Significance and Impact of the Study: The potential role of nitrite as an antimicrobial substance in the stomach may be of some importance in the ecology of the gastrointestinal tract and in host physiology.