The properties of aquatic dissolved organic matter (DOM) isolated by solid phase extraction (SPE) C-18 cartridges, ultrafiltration, and XAD chromatography are compared. Samples taken from the Suwannee River, Georgia, USA and McDonalds Branch in the Pine Barrens, New Jersey, USA were chosen to represent waters where DOM originates from predominantly terrestrially-derived (allochthonous) precursors. Pony Lake, Antarctica represented an exclusively algal/microbiallyderived (autochthonous) DOM. Fluorescence, UV absorption, 13 C NMR spectroscopy, and high-pressure size exclusion chromatography (HPSEC) were employed to discern differences and similarities between the DOM isolated by these three methods. Only subtle differences between isolation methods were observed for the terrestrially derived DOM samples when assayed by light and Aquat. Sci. 67 (2005) 61-71Aquatic Sciences fluorescence spectroscopy. Conversely, the Pony Lake DOM isolates exhibit greater variability when analyzed by these methods. 13 C-NMR analyses showed structural differences between the methods for all samples. HPSEC analysis also revealed differences with the C-18 isolates exhibiting the highest molecular weights. Thus, it appears that each method isolates sufficiently different fractions of DOM that can only be delineated when a consortium of analytical methods are used to assay the samples. Nonetheless real differences between autochthonous and allochthonous derived DOM were observed with the algal-derived samples exhibiting high fluorescence ratios and lower aromaticity relative to the terrestrially derived materials. These results demonstrate that caution must be exercised when interpreting DOM reactivity data that rely upon the use of specific fractions.