Dissolved organic carbon (DOC) flux from the Penobscot River and its major tributaries in Maine was determined using continuous discharge measurements, discrete water sampling, and the LOADEST regression software. The average daily flux during 2004–2007 was 71 kg C ha−1 yr−1 (392 Mt C d−1), an amount larger than measured in most northern temperate and boreal rivers. Distinct seasonal variation was observed in the relation between concentration and discharge (C–Q). During June through December (summer/fall), there was a relatively steep positive C–Q relation where concentration increased by a factor of 2–3 over the approximately 20-fold range of observed stream discharge for the Penobscot River near Eddington, Maine. In contrast, during January through May (winter/spring), DOC concentration did not increase with increasing discharge. In addition, we observed a major shift in the C–Q between 2004–2005 and 2006–2007, apparently resulting from unprecedented rainfall, runoff, and soil flushing beginning in late fall 2005. The relative contribution to the total Penobscot River basin DOC flux from each tributary varied dramatically by season, reflecting the role of large regulated reservoirs in certain basins. DOC concentration and flux per unit watershed area were highest in tributaries containing the largest areas in palustrine wetlands. Tributary DOC concentration and flux was positively correlated to percentage wetland area. Climatic or environmental changes that influence the magnitude or timing of river discharge or the abundance of wetlands will likely affect the export of DOC to the near-coastal ocean.