Nanobodies represent an interesting class of probes for the generic development of molecular imaging agents. We studied the relationship between tumor uptake of the epidermal growth factor receptor (EGFR)-specific nanobody (99m)Tc-7C12 and tumor burden and evaluated the possibility of using this probe to monitor tumor response to erlotinib. The specificity and affinity of (99m)Tc-7C12 was determined on A431 cells. Cells expressing firefly luciferase were used to evaluate tumor burden using bioluminescence imaging. We evaluated the effect of erlotinib on tumor burden and (99m)Tc-7C12 uptake in vitro as well as in vivo. In vivo bioluminescence imaging was performed followed by pinhole single-photon emission computed tomography/micro-computed tomography. (99m)Tc-7C12 binds specifically to the receptor with high affinity (3.67 +/- 0.59 nM). Erlotinib reduced tumor uptake and cell viability in a concentration-dependent manner. Tumor uptake of (99m)Tc-7C12 showed good correlation with tumor burden. Erlotinib treatment resulted in a progressive reduction of tumor burden and tumor uptake of (99m)Tc-7C12. (99m)Tc-7C12 binds to EGFR with high affinity and specificity. Tumor uptake is correlated with tumor burden. Quantification of (99m)Tc-7C12 uptake is promising for monitoring therapy response of EGFR-expressing tumors.