The complete genome of the gram-negative bacterial pathogenHelicobacter pylori, an important etiological agent of gastroduodenal disease in humans, has recently been published. This sequence revealed that the putative products of roughly one-third of the open reading frames (ORFs) have no significant homology to any known proteins. To be able to analyze the functions of all ORFs, we constructed an integration plasmid for H. pylori and used it to generate a random mutant library in this organism. This integration plasmid, designated pBCα3, integrated randomly into the chromosome of H. pylori. To test the capacity of this library to identify virulence genes, subsets of this library were screened for urease-negative mutants and for nonmotile mutants. Three urease-negative mutants in a subset of 1,251 mutants (0.25%) and 5 nonmotile mutants in a subset of 180 mutants (2.7%) were identified. Analysis of the disrupted ORFs in the urease-negative mutants revealed that two had disruptions of genes of the urease locus, ureBand ureI, and the third had a disruption of a unrelated gene; a homologue of deaD, which encodes an RNA helicase. Analysis of the disrupted ORFs in the nonmotile mutants revealed one ORF encoding a homologue of the paralyzed flagellar protein, previously shown to be involved in motility in Campylobacter jejuni. The other four ORFs have not been implicated in motility before. Based on these data, we concluded that we have generated a random insertion library in H. pylori that allows for the functional identification of genes in H. pylori.