Isotopic and chemical analyses were performed on crustaceans, forage fish, top predator fish, and sediment cores from Lake Ontario and two boreal forest lakes to investigate fractionation of the stable isotopes of Hg in aquatic ecosystems. Multicollector inductively coupled mass spectrometry was used to determine Hg isotope abundances. The Hg isotope data for all three lakes showed mass-independent variation in the organisms but only mass-dependent variation in the sediments. The mass-independent isotope effect was characterised by (1) selective enrichment in isotopes of odd mass number (199Hg and 201Hg), (2) enrichment in 201Hg relative to 199Hg, (3) an inverse relationship between isotopes of odd and even mass number in fish, and (4) a positive correlation with methylHg (CH3Hg+) concentration, and hence with trophic level (although lake whitefish were consistently anomalous, possibly owing to biochemical demethylation). Isotope signatures of species at the same trophic level varied with habitat and diet, differentiating between planktonic and benthic crustaceans and their predators, and between fish that frequent deep, cold water and fish of similar diet that prefer warmer, shallower water, because of corresponding differences in CH3Hg+ and inorganic Hg content. Isotopic analysis of CH3Hg+ and inorganic Hg extracted from lake trout proved that the mass-independent isotope effect was due to anomalously high abundances of 199Hg and 201Hg in CH3Hg+, as implied by the data for whole organisms, suggesting mass-independent fractionation during microbial methylation of Hg. The purely mass-dependent variation in the sediments is attributable to the fact that Hg in sediments is mostly inorganic. The mass-independent fractionation of Hg isotopes can be explained by effects of nuclear spin or nuclear field shift, or both, and penetration of the inner electron shells of Hg by valence electrons of Hg-binding ligands. The results of the research demonstrate that isotopic analysis of Hg could yield valuable information about the biogeochemical cycling of Hg.