Several nations have adopted hardness-modified copper (Cu) guidelines for protecting freshwater biota. However, there is a lack of good quality data and mechanistic understanding on the effects of true water hardness (calcium (Ca) and magnesium (Mg)) on the bioavailability and toxicity of Cu to freshwater biota, particularly macrophytes. This study determined the effect of true water hardness (35, 90 and 335 mg CaCO3/l, added as Ca and Mg chloride in a 1:1 mole ratio) on the cell surface binding affinity (log K), accumulation and toxicity (96 h growth (biomass and stem length) and photosynthetic pigment inhibition) of Cu in the free-floating submerged macrophyte, Ceratophyllum demersum, in a synthetic freshwater with constant alkalinity (16 mg CaCO3/l) and pH (7.0). There were no significant (P > 0.05) differences in the cell surface binding affinity, accumulation or toxicity of Cu in C. demersum with a 10-fold increase in water hardness from 35 to 335 mg CaCO3/l. The mean 96 h EC50 values (and 95% confidence intervals) for biomass, the most sensitive endpoint, were 8.4 (7.6-9.2), 8.9 (8.0-9.8) and 9.9 (9.1-10.7) μg/l Cu for 35, 90 and 335 mg CaCO3/l, respectively. Speciation calculations indicated only very small (1-6%) differences in the percentage distribution (i.e. bioavailability) of Cu over the hardness range. These collective results indicate no apparent competition between Cu and Ca/Mg for binding sites on the cell surface. Given that the mechanism of Cu uptake (via Cu-specific and Na-linked transporters) is fundamentally different to that of Cd, Ni, Pb and Zn (via Ca transporters), for which other hardness-dependent algorithms have been developed, it is doubtful whether a hardness-modified Cu guideline value will be sufficiently protective of sensitive freshwater biota, such as C. demersum, particularly in medium-hard fresh surface waters with low levels of dissolved organic carbon. The biotic ligand model offers a more flexible and mechanistic approach for deriving site-specific Cu (metal) guidelines for protecting freshwater biota.