Vacuolating cytotoxin (vacA) alleles of Helicobacter pylori vary, particularly in their mid region (which may be type m1 or m2) and their signal peptide coding region (type s1 or s2). We investigated nucleotide diversity among vacA alleles in strains from several locales in Asia, South America, and the USA. Phylogenetic analysis of vacA mid region sequences from 18 strains validated the division into two main groups (m1 and m2) and showed further significant divisions within these groups. Informative site analysis demonstrated one example of recombination between m1 and m2 alleles, and several examples of recombination among alleles within these groups. Recombination was not sufficiently extensive to destroy phylogenetic structure entirely. Synonymous nucleotide substitution rates were markedly different between regions of vacA, suggesting different evolutionary divergence times and implying horizontal transfer of genetic elements within vacA. Non-synonymous/ synonymous rate ratios were greater between m1 and m2 sequences than among m1 sequences, consistent with m1 and m2 alleles encoding functions fitting strains for slightly different ecological niches.The Gram-negative bacterium Helicobacter pylori is centrally involved in the pathogenesis of human peptic ulceration and is a major risk factor for the development of distal gastric adenocarcinoma and gastric lymphoma [4]. It exhibits an unusually high level of genetic diversity between strains [7], which has important implications for virulence, antibiotic resistance, and vaccine development. Implications of diversity for virulence are most obvious for the vacuolating cytotoxin gene, vacA, in that the various allelic types of vacA are differently associated with cytotoxicity and with disease state [1,2]. How genetic diversity in H. pylori is established and maintained remains unclear: recombination between strains appears important, but two studies using multilocus enzyme electrophoresis have given different assessments of [7,8]. Recently, Suerbaum et al. analyzed nucleotide sequence from parts of the two flagella genes flaA and flaB and from a small part of the vacuolating cytotoxin gene, vacA [24]: they found that recombination had been so frequent that all evidence of phylogenetic descent was obscured. We have studied vacA and have developed a pathogenically relevant PCR-based typing system based on vacA nucleotide sequence heterogeneity [1,2,26]. During the development of this system, we collected multiple sequence data from the two variable regions of vacA on which our typing system was based, the 0.7-kb mid region (for which we described two types, m1 and m2) and the region encoding the second half of the signal sequence (type s1a, s1b, s1c, or s2). Analysis of our sequence data provides contrasting results to those of Suerbaum et al. in that, although we demonstrate (by different methodology) that recombination has occurred between vacA alleles, we also show that this has not entirely disrupted the underlying phylogenetic relationship between se...