The bulk of biomedical positron emission tomography (PET)-scanning experiments are performed on mammals (i.e., rodents, pigs and dogs), and the technique is only infrequently applied to answer research questions in ectothermic vertebrates such as fish, amphibians and reptiles. Nevertheless, the many unique and interesting physiological characteristics in these ectothermic vertebrates could be addressed in detail through PET. The low metabolic rate of ectothermic animals, however, may compromise the validity of physiological and biochemical parameters derived from the images created by PET and other scanning modalities. Here, we review some of the considerations that should be taken into account when PET scanning fish, amphibians, and reptiles. We present specific results from our own experiments, many of which remain previously unpublished, and we draw on examples from the literature. We conclude that knowledge on the natural history and physiology of the species studied and an understanding of the limitations of the PET scanning techniques are necessary to avoid the design of faulty experiments and erroneous conclusions.