Radionuclide molecular imaging of epidermal growth factor receptor (EGFR) expression might permit the selection of patients for EGFR-targeting therapies. Designed ankyrin repeat protein (DARPin) E01 with a high affinity to the ectodomain III of the EGFR is a possible EGFR imaging probe. The goal of this study was to evaluate the potential of radiolabeled DARPin E01 for in vivo imaging of EGFR. DARPin E01 containing the (HE)-tag was site-specifically labeled with a residualizing Tc (using Tc]Tc(CO)). Two methods providing non-residualizing I labels, direct electrophilic radioiodination and indirect radioiodination using [I]I--iodobenzoate (PIB), were tested. [Tc]Tc-(HE)-E01 and [I]I-(HE)-E01-PIB preserved specific binding to EGFR-expressing cells and affinity in the single-digit nanomolar range. Direct labeling with I resulted in a substantial loss of binding. In vitro cellular processing studies showed that both [Tc]Tc-(HE)-E01 and [I]I-(HE)-E01-PIB had rapid binding and relatively slow internalization. Evaluation of [Tc]Tc-(HE)-E01 biodistribution in normal CD1 mice showed that its hepatic uptake was non-saturable, suggesting that this tracer does not bind to murine EGFR. A side-by-side comparison of biodistribution and tumor targeting of [Tc]Tc-(HE)-E01 and [I]I-(HE)-E01-PIB was performed in Nu/j mice bearing EGFR-positive A-431 and EGFR-negative Ramos human cancer xenografts. Both radiolabeled DARPins demonstrated EGFR-specific tumor uptake. However, [I]I-(HE)-E01-PIB had appreciably lower uptake in normal organs compared to [Tc]Tc-(HE)-E01, which provided significantly (