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To determine if there is any synergistic antitumor effect of ultrasound (US) in the presence of new quinolone (NQ) antibiotics, 0.2 mM solutions of lomefloxacin hydrochloride (LFLX), sparfloxacin (SPFX), ciprofloxacin hydrochloride (CPFX), and gatifloxacin hydrate (GFLX) were tested as sonodynamic agents against sarcoma 180 cells in vitro. After US irradiation at 2 W/cm2 for 30 and 60 s, the survival rates of tumor cells in the presence of NQ antibiotics were significantly lower than those in their absence (P<0.001). In May-Giemsa smears, most of the tumor cells remained intact in the control group. However, in the 0.2 mM SPFX group, the tumor cells were mostly fragmented. The synergistic antitumor effect of SPFX was dose-dependent. Furthermore, when D-mannitol was used with SPFX, the survival rate of tumor cells after irradiation was comparable with that when SPFX alone was applied, but when L-histidine was used concurrently, the survival rate of tumor cells was significantly higher than that when SPFX alone was applied. These findings suggest that NQ antibiotics would exhibit useful antitumor activity under US irradiation, and that generation of singlet oxygen is involved in the process of cell damage.