Carbon isotopes (13C and 14C) were used to study the characteristics and origin of humic substances in water samples from Lake Biwa in Japan. Humic substances were isolated from surface lake waters in each season by the XAD extraction method and their carbon isotopic ratios were measured by accelerator mass spectrometry. The Δ14C values of Biwa humic and fulvic acids indicate that these substances consist mainly of pre-bomb 14C. There is a negative correlation between Δ14C and δ13C values for the Biwa fulvic acids. The fulvic acid consists of younger organic materials with lower δ13C value in January, but older organics with higher δ13C in April and October. The major part of fulvic acid may be supplied from soil solution and/or groundwater in the watershed and their contribution varies with season.