A novel, promising strategy for cancer diagnosis and therapy is the use of a pretargeting approach. For this purpose, the non-natural DNA/RNA analogues Peptide Nucleic Acids (PNAs) are ideal candidates as in vivo recognition units due to their high metabolic stability and lack of unspecific accumulation. In the pretargeting approach, an unlabeled, highly specific antibody-PNA conjugate has sufficient time to target a tumor before administration of a small fast-clearing radiolabeled complementary PNA that hybridizes with the antibody-PNA conjugate at the tumor site. Herein, we report the first successful application of this multistep process using a PNA-modified epidermal growth factor receptor (EGFR) specific antibody (Cetuximab) and a complementary 99mTc-labeled PNA. In vivo studies on tumor bearing mice demonstrated a rapid and efficient in vivo hybridization of the radiolabeled PNA with the antibody-PNA conjugate. Decisively, a high specific tumor accumulation was observed with a tumor-to-muscle ratio of >8, resulting in a clear visualization of the tumor by single photon emission computed tomography (SPECT).