The use of small animal imaging is increasing in biomedical research thanks to its ability to localize altered biochemical and physiological processes in the living animal and to follow these processes longitudinally and noninvasively. In contrast to human studies, however, imaging of small animals generally requires anesthesia, and anesthetic agents can have unintended effects on animal physiology that may confound the results of the imaging studies. In addition, repeated anesthesia, animal preparation for imaging, exposure to ionizing radiation, and the administration of contrast agents may affect the processes under study. We discuss this interplay of factors for small animal imaging in the context of four common imaging modalities for small animals: positron emission tomography (PET) and single photon emission computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), and optical imaging. We discuss animal preparation for imaging, including choice of animal strain and gender, the role of fasting and diet, and the circadian cycle. We review common anesthesias used in small animal imaging, such as pentobarbital, ketamine/xylazine, and isoflurane, and describe techniques for monitoring the respiration and circulation of anesthetized animals that are being imaged as well as developments for imaging conscious animals. We present current imaging literature exemplifying how anesthesia and animal handling can influence the biodistribution of PET tracers. Finally, we discuss how longitudinal imaging studies may affect animals due to repeated injections of radioactivity or other substrates and the general effect of stress on the animals. In conclusion, there are many animal handling issues to consider when designing an imaging experiment. Reproducible experimental conditions require clear, consistent reporting, in the study design and throughout the experiment, of the animal strain and gender, fasting, anesthesia, and how often individual animals were imaged.