Camelidae possess an unusual class of antibodies devoid of light chains. Nanobodies are intact antigen-binding fragments that are stable, easily generated against different targets, and fully functional. Their rapid clearance from the blood circulation favors their use as imaging agents. We compared the in vivo tumor uptake and biodistribution of 2 anti-epidermal growth factor receptor (anti-EGFR) Nanobodies, Tc-99m-7C12 and Tc-99m-7D12. Methods: Nanobodies were labeled via their hexahistidine tail with Tc-99m-tricarbonyl (Tc-99m(CO)3) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFR-negative) xenografts were intravenously injected with Tc-99m-7C12 and Tc-99m-7D12 on separate days. Pinhole SPECT/micro-CT images were acquired at 1 h after administration to assess noninvasively the biodistribution and tumor targeting of the labeled compounds. Pinhole SPECT and micro-CT images from the same mouse were automatically fused on the basis of a mathematic rigid-body-transformation algorithm using Six Co-57 sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm(3)) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either Tc-99m-7C12 or Tc-99m-7D12 was also assessed; activity in the tumor and organs was recorded and expressed as percentage injected activity per gram (%IA/g). Results: Binding of both tracers was receptor-specific. Image analysis showed high and similar tumor uptake values for both Tc-99m-7C12 and Tc-99m-7D12 (4.55 +/- 0.24 %IA/cm(3) and 4.62 +/- 0.36 %IA/cm(3), respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16 +/- 0.14 for Tc-99m-7C12 and 1.49 +/- 0.60 for Tc-99m-7D12). Tc-99m-7C12 showed significantly higher kidney uptake (63.48 +/- 2.36 vs. 56.25 +/- 2.46 %IA/cm(3)) and lower liver uptake (2.55 +/- 0.26 vs. 4.88 +/- 0.86 %IA/cm(3)) than did Tc-99m-7D12. The ex vivo analysis confirmed the image quantification with high tumor-to-background ratio; however, Tc-99m-7C12 showed higher tumor uptake (9.11 +/- 1.12 %IA/g) than did Tc-99m-7D12 (6.09 +/- 0.77 %IA/g). Tc-99m-7D12 demonstrated significantly higher blood activity than did Tc-99m-7C12, but both showed short plasma half-lives (<10 min). Conclusion: The Nanobody fragments used here show high tumor uptake, low liver uptake, and rapid blood clearance. Nanobodies are promising probes for noninvasive radioimmunodetection of specific targets early after administration. On the basis of its favorable biodistribution, Tc-99m-7C12 was selected for further studies.