Nitrite may be generated by bacteria in urine during urinary tract infections. Acidification of nitrite results in the formation of nitric oxide (NO) and other reactive nitrogen oxides, which are toxic to a variety of microorganisms. We have studied NO formation and bacterial growth in mildly acidified human urine containing nitrite and the reducing agent vitamin C. Urine collected from healthy subjects was incubated in closed syringes at different pH values with varying amounts of nitrite and/or ascorbic acid added. NO generation was measured in headspace gas using a chemiluminescence technique. A similar setup was also used to study the growth of three strains of bacteria in urine. Mildly acidified nitrite-containing urine generated large amounts of NO and this production was greatly potentiated by ascorbic acid. The growth of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus saprophyticus was markedly reduced by the addition of nitrite to acidified urine. This inhibition was enhanced by ascorbic acid. In conclusion, we show that the growth of three common urinary pathogens is markedly inhibited in mildly acidified urine when nitrite is present. The bacteriostatic effect of acidified nitrite is likely related to the release of NO and other toxic reactive nitrogen intermediates. These results may help to explain the well-known beneficial effects of urinary acidification with, e.g., vitamin C in treatment and prevention of urinary tract infection.