South America (SA) contains Earth’s highest mammalian species richness, but this richness was exponentially higher just 10k years ago, prior to the last ice age extinction that eliminated more than 80% of mammals in the continent. With only two living genera restricted to tree-dwelling in rainforests, sloths once were a dominant group within SA mammalian ecosystems. Fossil sloths are generally considered plant eaters, like their modern relatives, but their extremely high diversity (in terms of body mass [4-4000 kg], geographical distribution [Patagonia to Alaska], habitats [coast, mountains, rainforest] and inferred locomotion [terrestrial, fossorial, arboreal, semiaquatic]) suggests that these were ecologically more versatile than traditionally thought. The primary goal of this project is to determine the dietary composition of fossil sloths assessing evidence for carnivory and the consumption of proteins of animal origin by means of amino acid compound specific isotope analyses, as suggested by our preliminary results. Since xenarthrans (the mammal group including sloths, armadillos, and anteaters) represented 1/3 of overall species diversity in South American mammal communities until as recently as 10,000 years ago, a discovery that fossil sloths possessed ecologies differing from those traditionally attributed to them could radically alter understanding of the entire community structure of terrestrial fossil mammals on the continent through time. Stable isotope analyses represent powerful, morphology-independent tools to directly reconstruct animal ecologies, and to test this controversial hypothesis of carnivory. To confidently apply this technique, however, several underlying assumptions first need to be tested on modern sloths and other mammals. Thus, the study will also test key assumptions inherent within isotopic trophic studies, and thus it is broadly relevant to our understanding of the isotopic signals preserved in other fossil species and ecosystems.