Copper-64-labeled monoclonal antibodies (mAbs) have previously demonstrated unexpectedly effective tumor control in rodent models of cancer at relatively low tumor-absorbed radiation doses. This property has been associated with delivery platforms resulting in cellular internalization. The purpose of the present studies was to evaluate the in vitro internalization and in vivo distribution of a two-antibody model of 64Cu radioimmunotherapy (RIT) in the same cell and animal models of cancer. Biodistributions of an internalizing antibody, cBR96, and a noninternalizing antibody, cT84.66, labeled with 64Cu, were obtained in nude mice bearing LS174T colon carcinoma xenografts from 15 min to 48 h. The 64Cu-DOTA-cBR96 conjugate demonstrated rapid tumor uptake, reaching 20.2% ID/g at 3 h and peaking at 35.4% ID/g by 24 h. Tumor accumulation of 64Cu-DOTA-cT84.66 was more gradual, 8.19% ID/g at 3 h and 43.8% ID/g by 24 h, but maximum uptake was not statistically different from 64Cu-DOTA-cBR96. Mouse xenograft dosimetry was estimated to be 1128 rad/mCi (304.9 mGy/MBq) for 64Cu-DOTA-cBR96 and 1409 rad/mCi (380.5 mGy/MBq) for 64Cu-DOTA-cT84.66. In LS174T cells, internalized radioactivity increased by a factor of 3.8 over 4 h for 64Cu-DOTA-cBR96, but remained unchanged 64Cu-DOTA-cT84.66. When normalized to uptake at 1 h, cellular efflux of 64Cu was essentially identical for both mAbs. The biodistributions and tumor dosimetry of these internalizing and noninternalizing radiolabeled mAbs were sufficiently similar for direct comparison of the therapeutic efficacies of low doses of 64Cu RIT agents in the same animal model of cancer.