PET allows non-invasive, quantitative and repetitive imaging of biological function in living animals. Small animal PET imaging with [18F]FDG has been successfully applied to investigation of metabolism, receptor–ligand interactions, gene expression, adoptive cell therapy and somatic gene therapy. Experimental condition of animal handling impacts on the biodistribution of [18F]FDG in small animal study. The small animal PET and CT images were registered using the hardware fiducial markers and small animal contour point. Tumor imaging in small animal with small animal [18F]FDG PET should be considered fasting, warming, and isoflurane anesthesia level. Registered imaging with small animal PET and CT image could be useful for the detection of tumor. Small animal experimental condition of animal handling and registration method will be of most importance for small lesion detection of metastases tumor model. (Nucl Med Mol Imaging 2008;42(1):1-7)