Natural organic matter (NOM) from two sites in South Australia were separated by Amicon YM and YC ultrafiltration (UF) membranes into five nominal fractions (<0.5, 0.5-3, 3-10, 10-30 and >30 kDa). These nominal fractions were then characterized for size and molecular weight (MW) distributions using flow field-flow fractionation. The results show that separation by UF did not produce fractions with the expected MW and size. Electrophoretic mobility measurements of the NOM fractions adsorbed to colloidal goethite showed no significant difference between the fractions. However solid-state (13)C NMR of the NOM fractions showed that the separation was influenced by molecular structure as well as molecular size. The results suggest that great caution needs to be exercised when interpreting molecular size and speciation results for humic substances obtained by membrane UF.