The feasibility of small animal imaging using a clinical positron emission tomography/computed tomography (PET/CT) scanner with [F-18]-fluoro-2-deoxy-D-glucose (FDG) was evaluated. As tumor-bearing small animal models, rabbits with VX-2 liver tumors, rats with mammary tumors on the back, and mice with LS174T human colon tumor xenografts were prepared. Two-dimensional PET, CT, and fused PET/CT images were obtained and reconstructed with a combined PET/CT system using a conventional protocol for humans and dedicated high-resolution mode protocols specialized for each species. Estimated radioactivity concentrations in tumors and normal organs determined noninvasively on FDG-PET/CT were compared with the actual tissue radioactivity levels determined from gamma-counting after vivisection in rats. In addition, recovery-corrected radioactivity concentrations were calculated and evaluated using the tumor/normal organ sizes measured on CT. Tumors in rabbits and rats were clearly visualized by FDG-PET/CT in the dedicated protocols, and images were considered suitable for research purposes. With the aid of thin-slice CT-mapping images, FDG uptake was correctly localized in the viable tumor regions. In mice, increased FDG uptake in tumors with varying activity levels was observed, but detailed anatomical information was not optimally provided from the images, even using specialized protocols. The estimated radioactivity concentrations of tumors and normal organs were close to the actual radioactivity concentrations obtained by gamma-counting (r = 0.97, P < 0.001, the estimated/actual slope: 1) when recovery correction was applied using the sample sizes measured on CT. FDG-PET/CT imaging with a modern clinical scanner was demonstrated to be feasible, of excellent quality, and quite quantitatively accurate for research in rabbits or rats with tumors of appropriate size (>2 cm without recovery correction and >1 cm with recovery correction). Evaluation of FDG uptake within a tumor was possible with the aid of CT images. Dedicated small animal PET/CT scanner would be better suited for evaluating tumor-bearing mice and likely could enhance imaging smaller tumors in rabbits or rats. Although it has limitations, small animal imaging with a clinical PET/CT scanner may be quite adequate for sequential noninvasive imaging in oncology research because the CT is of high resolution, allowing for localization of PET findings and for more precise noninvasive estimation of radioactivity concentration through partial volume corrections.