Background: Although the prevalence of Helicobacter pylori infection has been assessed in the National Health and Nutritional Examination Survey (NHANES), its possible relation with smoking has not been fully explored. Methods: We used the 1999-2000 NHANES data to examine whether smoking affects the prevalence of H. pylori infection in adults (n = 3689). The prevalence of H. pylori infection, as assessed by anti-H. pylori IgG enzyme-linked immunoassay, was computed according to smoking history and levels of serum cotinine. Results: Current smoking was associated with an increased prevalence of H. pylori infection (prevalence odds ratio =1.9; 95% confidence interval = 1.4-2.5) after controlling for possible confounders. Current smoking, as measured by increased serum cotinine, was also associated with an increased prevalence of H. pylori infection (1.6; 1.3-2.0). There was no evidence of a dose-response relation. Conclusions: Smoking may contribute to the persistence of H. pylori infection.