A Shigella flexneri 2a strain, which did not respond clinically or in laboratory tests to treatment with new quinolone derivatives, was isolated for the first time in Japan from a patient admitted for diarrhea to a Tokyo hospital. The mechanism of resistance was examined by sequencing the quinolone resistance determining region (QRDR) of the gyrA (a quinolone target enzyme) gene and by comparing the active efflux mechanisms of two strains isolated from this patient (before hospitalization and after tosufloxacin treatment) and one strain isolated from a patient with secondary infection with that of the standard strain ATCC 29903. DNA sequencing revealed that two amino acid substitutions, namely, Ser (TCG)-83-->Leu (TTG) and Asp (GAC)-87-->Gly (GGC), had occurred in the gyrA of all 3 strains isolated from the patients. Examination of the accumulation of antibiotics in these 3 strains revealed that the strain isolated after tosufloxacin treatment had the highest resistance to tosufloxacin, and exhibited decreased accumulation of tosufloxacin in the bacterial cells discharged after 5 days of treatment with this antibiotic. However, accumulation was restored by addition of a proton-pump inhibitor. These results suggest that the strains isolated from the inpatient and the patient with secondary infection acquired resistance due to dual gyrA mutation induced by treatment with new quinolone antibiotics. Furthermore, in addition to this dual mutation, the active efflux mechanism also appears to be associated with resistance in bacteria that have been exposed to tosufloxacin.