Abstract Two-dimensional heteronuclear multiple quantum coherence (HMQC) 1H-NMR was successfully applied to structural investigations of aquatic humic substance (HS) fractions obtained from multistage ultrafiltration (MST-UF) of a selected bog water HS (HO13-XAD8, German Research Program DFG-ROSIG). Details of the HMQC 1H-NMR novel in HS characterization are presented. The results of the combined procedure point out that structural units in isolated HS can be enriched in different mass fractions, for instance fatty acids in the fraction 1kDa, amino acids in the 1050 kDa fraction and methyl groups attached to quaternary carbon atoms in the high molecular mass fraction 50 kDa.