Dichlorofluorescin (DCFH), a widely used fluorescent probe for reactive oxygen species (ROS) was decomposed completely and generated two distinct fluorescent products by photo-irradiation at 254 nm for 30 min. In the previous study, we had shown that one was dichlorofluorescein (DCF), a well known oxidized product of DCFH. In this study we investigated the other product and identified it as monochlorofluorescein (MCF) by H-1-NMR and fast atom bombardment/mass spectrum (FAB/MS) analyses. MCF was generated by photo-irradiation, but not by ROS. On the other hand, DCF was produced by both photo-irradiation and ROS. MCF showed similar fluorescent emission spectrum to DCF, however, its fluorescence intensity was more than that of DCE The kinetic study suggested that MCF was not generated from DCF but from monochlorofluorescin, which might be generated from DCFH by photo-irradiation.