To determine the role of urease in colonization of Helicobacter pylori, we constructed a Ure- mutant by allelic exchange mutagenesis and examined the characteristics of the Ure- mutant and its parental strain. We also evaluated whether both strains would be able to colonize the stomach of nude mice. Kmr transformants obtained showed the Ure- phenotype, and one transformant, HPT73, was established as a knock-out mutant of the ureB gene by Southern analysis. Except for the null activity of the urease, HPT73 showed no difference from its parental strain, CPY3401, in growth rate in liquid medium, production of cytotoxin, and motility. However, CPY3401 had more acid resistance than HPT73. Moreover, CPY3401, but not HPT73, was detected in the nude mouse stomach. In the presence of HRA, both CPY3401 and HPT73 colonized the stomachs of nude mice. In summary, H. pylori requires urease for colonization in vivo and its survival under the acidic conditions of the stomach.