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Measuring chemical tracers in tissues of marine predators provides insight into the prey consumed and the predator’s contaminant exposure. In this study, samples from Type C killer whales (Orcinus orca) biopsied in Antarctica were analyzed for chemical tracers (i.e., stable isotopes of carbon and nitrogen, fatty acids, and persistent organic pollutants [POPs]). Profiles of these individual tracers were very different from those of killer whale populations that have been studied in the eastern North and eastern Tropical Pacific. For example, δ13C and δ15N stable isotope values and most POP concentrations were significantly lower in the Antarctic population. In addition, multivariate statistical analyses of both fatty acid and POP profiles found distinctly different patterns for Antarctic Type C whales compared to those from whales in the other populations. Similar assays were conducted on four species of Antarctic marine fish considered potential prey for Type C killer whales. Results were consistent with a diet of fish for Type C whales, but other species (e.g., low trophic-level marine mammals or penguins) could not be eliminated as supplemental prey.