Organs and tissues of whitefish and trout from mercury (Hg)-polluted Lake Ontario were analyzed for Hg isotopes, methylmercury (CH Hg ), 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 Hg/ Hg, Hg/ Hg, Hg/ Hg, and Hg/ Hg ratios and by Δ-values representing effects of MIF on Hg/ Hg and Hg/ Hg 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 Δ Hg and Δ Hg for whitefish than for trout; 4) nonlinear correlation between δ Hg and δ Hg for whitefish but linear correlation for trout; 5) an inverse correlation between the δ Hg values and CH Hg concentrations of whitefish and trout; 6) an inverse correlation between the δ Hg/δ Hg and CH Hg /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 Δ Hg and the CH Hg /inorganic Hg ratio for trout liver, kidneys, and gut. Environ Toxicol Chem 2018;37:515-529. © 2017 SETAC.