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Small animal PET scanning with 18F-FDG is increasingly used in murine tumor model. Experimental condition of animal handling impacts on the biodistribution of 18F-FDG in mouse study. The aim of this study was to optimize experimental conditions of animal handling and registration method for imaging lung metastasis tumor by using 18F-FDG small animal PET and CT. The experimental conditions for 18F-FDG biodistribution and animal PET imaging were 20 plusmn 2 h fasting and warming kept at 30degC. Biodistribution, standard uptake value (SUV) image and glucose SUV (SUVG) of 18F-FDG were evaluated and blood glucose level was also measured. Female C57BL/6 mice were injected intravenously with 2 times 105 of B16-F10 melanoma cells which developed metastasis tumors in lung. In lung metastasis tumor model, 18F-FDG images was obtained in optimized anesthesia condition and registered with anatomical clinical CT image. The small animal PET and CT images were registered using the hardware fiducial markers and small animal contour point in both data sets to perform a point- based rigid registration. Isoflurane 0.5 condition was chosen for lung metastasis tumor imaging. Lung metastasis tumor was clearly visualized by 18F-FDG PET image with Iso 0.5 anesthesia. Tumor imaging in lung region with 18F-FDG small animal PET should be considered feeding, temperature, and especially choice and level of anesthetic for minimizing background radioactivity. Fused imaging with small animal PET and CT image could be useful for the detection of metastatic tumor. This method improves the quantitative accuracy and interpretation of the tracer.