Small-animal PET scanning with ¹⁸F-FDG is increasingly used in murine models of human diseases. However, the impact of dietary conditions, mode of anesthesia, and ambient temperature on the biodistribution of ¹⁸F-FDG in mice has not been systematically studied so far. The aim of this study was to determine how these factors affect assessment of tumor glucose use by ¹⁸F-FDG PET and to develop an imaging protocol that optimizes visualization of tumor xenografts. Groups of severe combined immunodeficient (SCID) mice were first imaged by microPET with free access to food, at room temperature (20°C), and no anesthesia during the uptake period (reference condition). Subsequently, the impact of (a) fasting for 8–12 h, (b) warming the animals with a heating pad (30°C), and (c) general anesthesia using isoflurane or ketamine/xylazine on the ¹⁸F-FDG biodistribution was evaluated. Subcutaneously implanted human A431 epidermoid carcinoma and U251 glioblastoma cells served as tumor models. Depending on the study conditions, ¹⁸F-FDG uptake by normal tissues varied 3-fold for skeletal muscle, 13-fold for brown adipose tissue, and 15-fold for myocardium. Warming and fasting significantly reduced the intense ¹⁸F-FDG uptake by brown adipose tissue observed under the reference condition and markedly improved visualization of tumor xenografts. Although tumor ¹⁸F-FDG uptake was not above background activity under the reference condition, tumors demonstrated marked focal ¹⁸F-FDG uptake in warmed and fasted animals. Quantitatively, tumor ¹⁸F-FDG uptake increased 4-fold and tumor-to-organ ratios were increased up to 17-fold. Ketamine/xylazine anesthesia caused marked hyperglycemia and was not further evaluated. Isoflurane anesthesia only mildly increased blood glucose levels and had no significant effect on tumor ¹⁸F-FDG uptake. Isoflurane markedly reduced ¹⁸F-FDG uptake by brown adipose tissue and skeletal muscle but increased the activity concentration in liver, myocardium, and kidney. Animal handling has a dramatic effect on ¹⁸F-FDG biodistribution and significantly influences the results of microPET studies in tumor-bearing mice. To improve tumor visualization mice should be fasted and warmed before ¹⁸F-FDG injection and during the uptake period. Isoflurane appears well suited for anesthesia of tumor-bearing mice, whereas ketamine/xylazine should be used with caution, as it may induce marked hyperglycemia.