Within the aquatic carbon cycle, dissolved organic carbon (DOC) plays key roles: as a link between terrestrial and aquatic systems, a major food source for biota, a player in photochemical reactions and a sunscreen/competitor for light for aquatic organisms. The molecular-level composition of the dissolved organic matter (DOM) is believed to determine the efficacy of DOM in these roles. While much detailed compositional information is available on selected DOM isolates from both freshwater and marine systems, relatively few studies have presented molecular information for multiple sites within the same system and few of these have focused upon large lakes. Therefore, we present an initial study of surface water DOM characteristics in a temperate large lake (Lake Superior, USA) and selected tributaries. UV-visible spectroscopy, electrospray mass spectrometry and Fourier transform infrared spectroscopy (FTIR) analyses of bulk DOM, along with FTIR and direct temperature-resolved mass spectrometry analyses of C18 extracts, illustrate a shift from higher molecular weight organic matter enriched in protein and lignin at the tributary sites toward compositionally different material in the open lake. Differences in optical characteristics and DOC concentration between tributary and open lake sites are consistent with possible photodegradation of tributary DOM. Comparison of our FTIR data with those from a previous Chesapeake Bay study indicates that DOM in both the freshwater tributaries and the open lake contain a wider range of aromatic and carbohydrate components than that in the saltwater tributary and bay. This highlights the need for further cross system comparisons of natural organic matter composition.