Aquatic humic (HA) and fulvic acids (FA) from two different origins were fractionated by the tandem size-exclusion chromatography and polyacrylamide gel electrophoresis (tandem SEC-PAGE). Three HA and two FA fractions exhibited different molecular sizes, electrophoretic mobilities and structural compositions based on elemental analyses and pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) after tetramethyl-ammonium hydroxide (TMAH) treatment. Based on comparison of the size-separates and unfractionated bulk samples, the highest molecular size fractions of aquatic humic substances (HS) appear to be enriched in fatty acids (mainly C 16 and C 18 ), which are strictly linked to the humic core. This and the presence of low chain fatty acids and unsaturated and di-tri acids may suggest a microbial rather than a vascular plant origin. Even if the majority of lignin derived components in aquatic HS represent relatively high state of biodegradation (oxidation), a certain variation between different sizefractions also exists, supporting a high heterogeneity and composite nature of aquatic HS. Although SEC-PAGE fractionation appears to produce fairly homogeneous HS fractions, the absorption of some urea into of the separates cannot be completely avoided in this method, which limits its suitability for evaluating, especially the presence of N-containing constituents.