A total of 22 type II restriction endonucleases with 18 distinct specificities have been identified in six Helicobacter pylori strains. Among these 18 specificities are three completely new endonucleases, Hpy178III, Hpy99I, and Hpy188I, that specifically cleave DNA at TC-NNGA, CGWCG, and TCNGA sites, respectively. The set of endonucleases identified in each strain varies, but all have four-or five-base recognition sequences. Among 16 H. pylori strains, examination of the DNA modification status at the recognition sites of 15 restriction endonucleases reveals that each strain has a substantially different complement of type II modification systems. We conclude that the type II restriction-modification systems in H. pylori are highly diverse between strains, a unique characteristic of H. pylori. The diverse methylation status of H. pylori chromosomal DNA may serve as a new typing system to discriminate H. pylori isolates for epidemiological and clinical purposes. This study also demonstrates that H. pylori is a rich source of type II restriction endonucleases.T ype II restriction-modification (R-M) systems are pairs of enzymes: one, a restriction endonuclease (RE); the other, a methyltransferase, with opposing intracellular activities (1, 2). The RE recognizes specific sequences in DNA and cleaves the DNA at a particular site whereas the cognate methyltransferase modifies DNA within the same recognition sequence, thereby preventing cleavage by the RE. By possessing these two opposing enzymes, bacteria may protect their own DNA and degrade foreign DNA, thus limiting the spread of invading DNA molecules within the bacterial population (3-6). In addition, DNA methylation may be involved in regulation of chromosomal DNA replication (7,8) and gene expression (9, 10), transposon movement (11), or DNA mismatch repair (12).Helicobacter pylori are bacteria that colonize the human stomach and increase the risk of development of peptic ulcer disease and gastric adenocarcinoma (13-15). Analysis of the entire genomic sequence of H. pylori strains 26695 and J99 predicted that they possess an unusually high number (14 or 15) of type II R-M systems (16,17). Comparison of these two strains showed that the two genomes are quite similar, with only 6-7% strain-specific genes (17); R-M systems are a major source of the strain differences. Furthermore, comparison of strains J166 and 26695 using a PCRbased subtractive hybridization method (18) showed that 7 of 18 J166-specific DNA clones had homology to the genes of R-M systems. In total, these studies demonstrated that, at the genomic level, R-M systems are quite diverse among the three strains examined. Whether this diversity is a general phenomenon among various H. pylori strains and which functional R-M systems are present in these strains remain as unanswered questions. Thus far, only the iceA-hpyIM locus in H. pylori, an ulcer-related NlaIII-like type II R-M system (19,20), has been studied among various strains, which indicates that the M gene is conserved whereas the R gene (ice...