The determination of H. pylori in vitro antibiotic susceptibility to the four key antibiotics and the underlying mechanisms of resistance were investigated. Levels of antibiotic resistance in two independent populations in England and Wales were determined. In the urban London-based population, metronidazole resistance was present in 59% and clarithromycin resistance was in 11% of the 101 H. pylori isolates. Of 363 isolates from rural Bangor, North Wales, 23% were metronidazole resistant and 7% were clarithromycin resistant. Combined clarithromycin and metronidazole resistance occurred in 9% of London and in 4% from Bangor isolates. Analysis of patient questionnaires completed at the London centres identified a significant association between metronidazole resistance and birth outside the UK. Analysis of the method used to determine metronidazole susceptibility status indicated that anaerobic pre-incubation gave results falsely indicating susceptibility. This study would recommend the use of Etest strips on 10% Mueller-Hinton blood agar, incubated microaerophilically at 37°C for 48 h. Comparison of detection methods for 23Sr rDNA mutations associated with H. pylori clarithromycin resistance showed a novel 3’-mismatched reverse primer PCR to be the most sensitive approach. Sequence analysis of 16S rDNA in tetracycline resistant isolates identified the A926g mutation for the first time from a UK H. pylori isolate and a novel A926C mutation. The mechanism of unstable amoxicillin resistance was investigated. Novel PCR-RFLP assays, exploiting sequence variability in the outer membrane protein genes hopB and hopC, indicated no association with antibiotic resistance, but have potential for use as a novel genotyping tool for H. pylori. This study highlights the need for H. pylori antibiotic resistance surveillance at the regional level to help guide treatment regimens.