Resistance to antibiotics, especially clarithromycin, is the major cause of the failure to eradicate Helicobacter pylori. There are few studies in children concerning fluoroquinolone activity against H. pylori. Primary resistance to antibiotics including fluoroquinolones was studied in 55 H. pylori strains isolated from Japanese children. DNA sequences of the gyrA gene in fluoroquinolone-resistant strains were determined. Twelve strains (21.8%) were resistant to clarithromycin and three (5.5%) were resistant to both levofloxacin and ciprofloxacin. Out of 12 clarithromycin-resistant strains, 11 (91.7%) were susceptible to levofloxacin and ciprofloxacin. Sequence analysis in three fluoroquinolone-resistant strains showed point mutations of the gyrA gene at G271A, G271T and A272G, indicating mutations of the codon Asp91 in the fluoroquinolone-resistance-determining region of the DNA gyrase. The results suggest that fluoroquinolones should be considered as an option for second- or third-line H. pylori eradication therapy in children.