This study investigated the concentration and isotopic composition of different size fractions of organic matter (OM) in Lake Superior and in one of its many tributary streams and rivers (Amity Creek, Duluth, Minnesota, USA). Structural compositional drivers of the δ14C of high molecular weight (HMW, >1kDa) dissolved organic carbon (DOC) in the lake were also evaluated. Low molecular weight (LMW, <1kDa) DOC was the fraction containing the largest proportion (68-88%) of organic carbon (OC) in the lake. Particulate organic carbon (POC, >0.7μm) was generally 13C-depleted (-29±1.2‰) relative to "bulk" (<0.7μm) DOC (-26.4±0.7‰), "init" (<0.2μm) DOC (-26.6±0.8‰), HMW DOC (-26.9±0.3‰) and LMW DOC (-26.5±0.9‰), and had more variable 14C content (δ14C of -94‰ to 53‰; 735years BP to modern) than the other size fractions. Init DOC (δ14C of 17-59‰), HMW DOC (δ14C of 23-64‰) and LMW DOC (δ14C of 16-62‰) all reflected contemporary (modern) radiocarbon signatures. Bulk DOC (δ14C of -19‰ to 57‰; 90years BP to modern) had modern radiocarbon values in the offshore sites (δ14C of 2-57‰) with pre-aged samples (δ14C of -8‰ to -19‰) seen at the nearshore site. HMW DOM was relatively N-poor (C:N of 12-19) compared to particulate organic matter (POM, C:N of 8-10) revealing either a more diagenetically altered state or contrasting sources. 13C NMR data showed that biochemical composition of HMW DOC in Lake Superior was dominated by carbohydrates (53-65%) with only trace aromatic components (2-4%). Structurally complex components such as heteropolysaccharides (HPS), amide/peptides and amino sugars (AMS) constitute 75-84% of HMW DOC whereas carboxylic-rich alicyclic molecules (CRAM) made up 16-25% of HMW DOC in the lake. Combined HPS and AMS, O-alkyl carbohydrate carbon, and total carbohydrate carbon contents were significantly positively correlated to the δ14C of HMW DOC suggesting they contribute a contemporary 14C-enriched component to the HMW DOC in the lake. In contrast, CRAM and aliphatic carbons were significantly inversely correlated with δ14C of HMW DOC implying that these represent 14C-depleted (older) components of HMW DOC in the lake. At Amity Creek, storm events led to larger loads of bulk DOC and POC, which were both 14C-enriched (modern). However, in baseflow conditions 14C-depleted bulk DOC and POC were exported. LMW DOC from the creek was considerably 14C-depleted compared to concurrent HMW DOC.