A procedure for separating aquatic humic substances into discrete molecular size fractions by the combined use of ultrafiltration and dialysis was tested. The molecular size distributions of separated fractions were examined by application of samples to a gel filtration chromatography column. Variations in the elution patterns observed for the separated fractions of small and intermediate molecular size were largely consistent with predicted effects of separation procedures. In contrast, humic substances of large molecular size were not separated effectively by dialysis using dialysis membranes of large molecular weight cut‐off. Small quantities of humic material of large molecular size were observed in most of the ultrafiltrates. This phenomenon may have been an artefact of filtration and/or the result of reconfiguration of material after separation.