Helicobacter pylori culture on gastric biopsy was performed on 4964 subjects aged <18 years from 1988 to 2007 at a central laboratory in Brussels. The total number of biopsies increased markedly from 941 in 1988-1993 to 1608 in 2004-2007. Biopsies were repeated at least once for 922 subjects (603 initially negative and 319 initially positive for H. pylori). Persistence rate of H. pylori at 1 year after initial positive biopsy was greater in the 1998-2007 cohort than in the 1988-1997 cohort (72.7% vs. 45.8%, P = 0.002), suggesting a tailored selection of candidates for biopsy with non-invasive tests (13C urea breath test). Of 68 subjects initially positive and re-examined subsequently after a documented cure, re-infection/relapse rate was 48.6% within 5 years post-elimination of H. pylori. Acquisition rate over 10 years follow-up in the initially negative cohort (603 patients) was 38.7% (re-infection/relapse vs. acquisition: P < 0.001). Multivariate analysis showed a fourfold greater risk of H. pylori acquisition in children of non-European origin vs. European origin (P < 0.001). Clarithromycin and metronidazole susceptibility were determined in 226 and 223 paired positive cultures in cases of re-infection/relapse or persistence. An initial non-susceptibility profile was highly predictive of a subsequent non-susceptibility profile, and the non-susceptible proportion increased markedly from 13.3% to 21.2% for clarithromycin (P < 0.001) and from 27.3% to 35.0% for metronidazole (P = 0.014), with no difference regarding European or non-European origin.