PURPOSE: Several investigators reported the polymerase chain reaction (PCR) method was more sensitive than culture or other routine laboratory tests for the detection of H. pylori. In this study, we established the nested PCR method for the sensitive and specific determination of H. pylori from paraffin-embedded gastric cancer samples, and the polymorphisms of H. pylori urease A gene were analyzed using by restriction fragment length polymorphism (RFLP). MATERIALS AND METHODS: It was subjected to the nested PCR using two primer pairs from the urease A gene of H. pylori. The sensitivity of the nested PCR assay was investigated with serial dilutions of positive DNA of H. pylori. Polymorphisms of H. pylori were determined by digestion of thirty six PCR positive products with five different restriction endonuclease-MspI, AluI, DdeI, BstNI, and HinfI. RESULTS: Amplified H. pylori PCR products were detected to 106 dilutions (10-3 fg) by nested PCR technique. The polymorphic patterns of five types of H. pylori were found by MspI, DdeI and AluI. Sequence of type V was confirmed by direct sequencing and the sequences recognized by BstNI and HinfI were conserved regions. CONCLUSIONS: Nested PCR technique is a accurate, sensitive and reliable method for the laboratory diagnosis of H. pylori infection. Moreover nested PCR-RFLP analysis has a potential to differentiate H. pylori strains.