Overexpression of epidermal growth factor receptor (EGFR) in several types of malignant tumors correlates with disease progression. EGFR could, therefore, be an excellent candidate for targeted radionuclide diagnostics. However, the high natural expression of EGFR in the liver may be problematic. The aim of this study was to improve the tumor-to-liver ratio of radiolabeled epidermal growth factor (EGF) by blocking its uptake by the liver with a nonradiolabeled EGFR-targeting molecule in tumor-bearing mice. Intraperitoneally injected nonradiolabeled EGF was first evaluated as a blocking agent, preadministered at various time intervals before intravenous injection of (125)I-labeled EGF. The anti-EGFR Affibody molecule (Z(EGFR:955))(2) was then assessed as a blocking agent of (111)In-labeled EGF in a dual isotope study (50, 100, and 200 microg, preadministered 30 or 60 min before (111)In-EGF). The 30-min preadministration of nonradiolabeled EGF significantly decreased (125)I-EGF uptake in the liver, whereas uptake in the tumor remained unchanged. Furthermore, preadministration of only 50 microg (Z(EGFR:955))(2) as a blocking agent 30 min before the (111)In-EGF decreased the uptake of (111)In-EGF by the liver and increased its uptake by the tumor, thereby increasing the tumor-to-liver ratio sixfold. We conclude that the Affibody molecule (Z(EGFR:955))(2) shows promise as a blocking agent that could enhance the outcome of radionuclide-based EGFR-expressing tumor diagnostics and imaging.