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For decades, plant ecologists have used naturally occurring stable isotope ratios to disentangle ecological and physiological processes. The methodology can also become a very powerful tool in animal ecology. However, the application of the technique relies on assumptions that are not widely recognized and that have been rarely tested. The purpose of this communication is to identify these assumptions, to characterize the conditions in which they are not met, and to suggest the laboratory experiments that are needed to validate them. The ease with which isotopic data can be gathered and the growing popularity of the method are generating a large amount of data on the isotopic ecology of animals. The proper interpretation of these data demands that we identify the assumptions on which these inferences are based, and that we conduct comparative laboratory experiments to assess their validity.