Photodegradation of dissolved organic matter (DOM), collected from two upstream and one downstream sites in the Lake Biwa watershed in Japan, was investigated using fluorescence properties of three-dimensional excitation emission matrix (3-D EEM). Solar irradiation caused a decrease in the dissolved organic carbon (DOC) concentration and in the fluorescence peak intensity (FI) of fulvic acid (FA)- and protein-like substances in DOM. Mineralization of DOC during an irradiation period (13 days) was observed to be higher in upstream samples (32-36%) compared to that in downstream sample (16%). DOC concentration in samples stored in the dark significantly decreased in the downstream sample (16%), while those in upstream samples hardly decreased (1-8%). The FI of FA-like substance decreased by 72-84% during 13-days irradiation (integrated solar intensity = 176 MJ m-2). The protein-like fluorescence was only observed in the downstream sample and the net decrease in the FI was 59% in the irradiated sample. The first-order reaction kinetics was applied to analyze the photodegradation of DOM. Two-step photodegradation was suggested for the fluorescent DOM (FDOM), while not for the DOC. Results obtained here suggested that FA-like FDOM is more susceptible to photodegradation compared with those of protein-like substances and DOC.