The association between Helicobacter pylori (H. pylori) and gastric adenocarcinoma remains controversial. However, the prevalence of H. pylori infection in gastric adenocarcinoma varies with the method of detection used, eg, serology, histology, culture, and polymerase chain reaction. However, studies on gastric adenocarcinoma by these methods remain inconclusive. We compared the results of serology, histology, and polymerase chain reaction for detection of H. pylori infection in 12 patients with gastric adenocarcinoma to investigate the actual status of H. pylori infection in gastric adenocarcinoma. IgG antibodies to H. pylori were examined in the sera, and histology and polymerase chain reaction were used for identification of H. pylori on the resected gastric tissues of 12 patients with gastric adenocarcinoma. Among them. H. pylori infection was verified by serology in eight, polymerase chain reaction in seven, and histology in five. In all positive cases, H. pylori was identified only in the non-tumorous part of the gastric tissue. In all four seronegative patients, H. pylori was not present in any of the tissues examined by histology and polymerase chain reaction. We conclude that: 1) polymerase chain reaction is more sensitive than histology; 2) a good correlation between serology and polymerase chain reaction is found; and 3) H. pylori infection is absent in a proportion of patients with gastric adenocarcinoma identified by these methods.