The present report describes a novel method for genotyping the virulence-associated vacA intermediate (i) region of Helicobacter pylori in archive material. vacA i-region genotypes as determined by the novel method were completely concordant with those of sequence analysis and with those of functional vacuolation activity. The method was further validated directly in gastric biopsy specimens of 386 H. pylori-positive cases, and effective characterization of the vacA i region was obtained in 191 of 192 (99.5%) frozen and in 186 of 194 (95.9%) formalin-fixed paraffin-embedded gastric biopsy specimens, respectively. The genotyping method was next used to address the relationship between the vacA genotypes and the cagA status. The vacA i1 genotype was associated with vacA s1 (where s indicates signal region), vacA m1 (where m indicates middle region), and cagA-positive genotypes (P < 0.0001), while the vacA i2 genotype was closely related with vacA s2, vacA m2, and cagA-negative genotypes (P < 0.0001). The relationship between H. pylori vacA i-region genotypes and gastric disease development was subsequently evaluated in the Portuguese population. Patients infected with vacA i1 strains showed an increased risk for gastric atrophy and for gastric carcinoma, with odds ratios of 8.0 (95% confidence interval [CI], 2.3 to 27) and of 22 (95% CI, 7.9 to 63), respectively. Taken together, the results show that this novel H. pylori vacA i-region genotyping method can be applied directly to archive material, providing a fast evaluation of strain virulence determinants without the need of culture. The results further emphasize that the characterization of the vacA i region may be useful to identify patients at higher risk of gastric carcinoma development.