Stable isotope analysis (SIA) has become a powerful tool for ecotoxicologists to study dietary exposure and biomagnification of contaminants in wild animal populations. The use of SIA in ecotoxicology continues to expand and, while much more is known about the mechanisms driving patterns of isotopic ratios in consumers, there remain several considerations or sources of uncertainty that can influence interpretation of data from field studies. We outline current uses of SIA in ecotoxicology, including estimating the importance of dietary sources of carbon and their application in biomagnification studies, and we present six main considerations or sources of uncertainty associated with the approach: (1) unequal diet-tissue stable isotope fractionation among species, (2) variable diet-tissue stable isotope fractionation within a given species, (3) different stable isotope ratios in different tissues of the animal, (4) fluctuating baseline stable isotope ratios across systems, (5) the presence of true omnivores, and (6) movement of animals and nutrients between food webs. Since these considerations or sources of uncertainty are difficult to assess in field studies, we advocate that researchers consider the following in designing ecotoxicological research and interpreting results: assess and utilize variation in stable isotope diet-tissue fractionation among animal groups available in the literature; determine stable isotope ratios in multiple tissues to provide a temporal assessment of feeding; adequately characterize baseline isotope ratios; utilize stomach contents when possible; and assess and integrate life history of study animals in a system.