Water samples were collected from 10 locations along major rivers of the Piracicaba River basin during 22 months. DOC concentrations, C and N elemental composition and carbon isotopic composition in the dissolved fraction were analyzed. Riverine organic matter origin was investigated based on specific biomarkers of possible sources (plants, soils, sewage, phytoplankton) and particulate fractions. DOC concentrations varied from 2.52 at Jaguari River to 4.93 mg.l-1 at Piracicaba River, both occurring during dry season. DOC fluxes were 1.57 in the Jaguari R., 1.73 in the Atibaia R. and 2.18 ton.km-2.year-1 in the Piracicaba R.. At less polluted sites, there was a higher influence from lignin phenols, mainly during the wet season, with the highest value of 7.59 mg.100mgOC-1 for the coarse fraction. The dissolved fraction had the lowest lignin concentrations and the fine fraction intermediary concentrations. The heaviest d13C values and the highest lignin concentrations during the wet season were related to soils from the drainage basin. The ultimate source of organic matter for soils appears to be a mixture from C3 and mainly C4 plants, since pasture and sugar cane predominate in the basin for the last 50 years. During the dry season, the sterol coprostanol (3b-Cholestan-ol) was found in high concentrations, indicating organic material loaded into the system from sewage. A value >0.7 for the ratio [coprostanol / (coprostanol + cholestanol)], indicative of fecal influence was found in all rivers samples. N-enriched and 13C depleted samples and high value of the ratio [cholesterol / (stigmasterol + sitosterol)], were related to phytoplankton growth. In spite of the alternation between terrestrial plants and phytoplankton as the main source for riverine organic matter during wet and dry season, respectively, the sewage is an important source to the Piracicaba River basin throughout the year.