The harsh chemical conditions involved in the isolation of fulvic acids (FA) and humic acids (HA) have been identified as a possible contributing factor to the significant mismatch between in situ measurements and model predictions of trace metal speciation in freshwaters, resulting from the use of isolated FA and HA in model calibration. A set of experimental assays were developed to enable Cu binding to DOM to be measured over the full range of [Cu]/[DOC] ratios (1–460 μmol g–1) observed in surface freshwaters. They were applied to the widely used and traditionally isolated Suwannee River HA and FA and to DOM isolated from headwater streams by a mild procedure using minimal chemical treatment. Good agreement was observed between measured free ion activities and those predicted using both WHAM/Model VII and NICA–Donnan speciation models for both traditionally and mildly isolated DOM. Agreement to within a factor of 2 for WHAM/Model VII contrasts with 100-fold differences previously reported between in situ Cu2+ measurements and model predictions for a wide range of conditions. The results demonstrate that (a) existing speciation models are capable of accurately predicting Cu-humic binding in natural waters at environmentally realistic [Cu]/[DOC] ratios, under equilibrium conditions, and (b) that the isolation procedures traditionally used for HA and FA do not appreciably affect their binding characteristics.