Abstract   Organs and tissues of whitefish and trout from mercury (Hg)-polluted Lake Ontario were analyzed for Hg isotopes, methylmercury (CH3Hg+), and inorganic Hg to investigate possible mass-dependent fractionation (MDF) and mass-independent fractionation (MIF) of Hg isotopes by physiological processes of the fish. Isotope signatures of different body parts were defined by δ-values of 198Hg/202Hg, 199Hg/202Hg, 200Hg/202Hg, and 201Hg/202Hg ratios and by Δ-values representing effects of MIF on 199Hg/202Hg and 201Hg/202Hg ratios. The research yielded the following evidence for MDF and MIF, including MIF of isotopes with even as well as odd mass numbers, by metabolic activities: 1) anomalously low δ-values for whitefish kidneys but not for trout kidneys; 2) widely varying differences between the δ-values of different body parts of whitefish but practically uniform differences for those of trout; 3) different relationships between Δ199Hg and Δ201Hg for whitefish than for trout; 4) nonlinear correlation between δ198Hg and δ200Hg for whitefish but linear correlation for trout; 5) an inverse correlation between the δ199Hg values and CH3Hg+ concentrations of whitefish and trout; 6) an inverse correlation between the δ201Hg/δ199Hg and CH3Hg+/inorganic Hg ratios of trout kidneys and gills (and lipids of trout near the sources of pollution) but a positive correlation for muscle, liver, and gut; and 7) inverse correlations between Δ199Hg and the CH3Hg+/inorganic Hg ratio for trout liver, kidneys, and gut. Environ Toxicol Chem 2018;37:515–529. © 2017 SETAC