Monoclonal antibodies (Mabs) with tumor specificity and 2-[18F]-fluoro-2-deoxy-D-glucose (FDG) are increasingly being used in cancer imaging. To better understand the mechanism and location of their uptake in tumors, nude mice bearing HTB77 IP3 ovarian cancer xenografts were injected intravenously with 3H-2-fluoro-2-deoxy-D-glucose (3H-DG) or 14C-2-deoxy-D-glucose (14C-DG), with 125I-5G6.4, a Mab that was developed against the HTB77 IP3 cell line (specific Mab) or with 125I-UPC-10 (nonspecific Mab). Tumors were excised at 2 hr or 6 days postinjection and autoradiography was performed. In tumors from animals receiving the Mabs, the grains were concentrated mainly on tumor cells near connective tissue ridges bearing blood vessels at 2 hr postinjection. At 6 days postinjection, further penetration into the tumors was seen, but four times more grains were seen on the viable cancer cells in the tumors receiving the specific Mab than in those receiving the irrelevant Mab. The selective accumulation of 2-DG in viable cancer cells and the negligible concentration of 2-DG in necrotic areas may be preferable to tumor-specific Mabs in assessing the extent of viable tumor and treatment response. Knowledge of the localization of a tracer at the microscopic level is essential to the understanding of the signal depicted on nuclear images using either type of radiotracer.