The 23S rRNA gene in clinical isolates of Helicobacter pylori isolated between 1995 and 2004 from Japan was investigated and the relationship between mutations in this gene and clarithromycin susceptibility was studied. Among nine mutations that have previously been reported to confer clarithromycin resistance, an adenine-->guanine transition at position 2142 (A2142G) or 2143 (A2143G) was detected in all clarithromycin-resistant strains (n=67) but not in any clarithromycin-susceptible strains (n=17). Mutations at positions 2182, 2223, 2244 and 2288 have previously been reported to confer clarithromycin resistance in H. pylori isolates from Bangladesh, China and Brazil. However, these mutations were not associated with clarithromycin resistance in H. pylori isolates from Japan in this study. Other mutations at positions 2115, 2144 and 2711, which have also been reported to confer clarithromycin resistance in H. pylori from Sweden and Italy, were not detected in the strains in this study. Our results suggest that susceptibility to clarithromycin is predicted by detection of mutations at positions 2142 and 2143 of the 23S rRNA gene in H. pylori isolates in Japan.