Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.