To elucidate the mechanism of phototoxicity induced as a side effect by some of the new quinolone antibiotics, we studied sparfloxacin (SPFX), lomefloxacin, enoxacin, ofloxacin, and ciprofloxacin. We first examined the photosensitized formation of reactive oxygen species such as singlet oxygen (1O2) and superoxide anion (O2-) mediated by the new quinolones. Although a large number of studies have been reported, there is no direct evidence that these drugs generate reactive oxygen species. We employed a near-infrared emission spectrometer to detect 1O2-specific emission (1268 nm), and the nitroblue tetrazolium reduction method to detect O2-. All the quinolones investigated in this study were found to produce 1O2. Four drugs, but not SPFX, produced O2-. We also examined photodynamic DNA strand-breaking activity as a possible mechanism to explain the participation of reactive oxygen species in the phototoxicity of the drugs. All the drugs exhibited photodynamic DNA strand-breaking activity. The inhibitory effect of scavengers of reactive oxygen species indicated that the main active species was 1O2. The DNA strand-breaking activity was correlated not with the 1O2-forming ability, but with the affinity of the drugs for DNA. This result may be due to the short lifetime of 1O2. These data suggested that the phototoxicity of the new quinolones was related to DNA damage caused by reactive oxygen species, especially 1O2.