Epidermal growth factor receptor (EGFR) is overexpressed in many carcinomas and remains a prime target for diagnostic and therapeutic applications. There is a need to develop noninvasive methods to identify the subset of patients that is most likely to benefit from EGFR-targeted treatment. Noninvasive imaging of EGFR messenger RNA (mRNA) expression may be a useful approach. The aim of this study was to develop a method for preparation of single-photon-emitting probes, Tc-99m-labeled EGFR mRNA antisense peptide nucleic acid (PNA) (Tc-99m-EGFR-PNA), and nontargeting control (Tc-99m-CTL-PNA) and to evaluate their feasibility for imaging EGFR mRNA overexpression in malignant tumors in vivo. Methods: On the 5' terminus of synthesized single-stranded 17-mer antisense EGFR mRNA antisense PNA and mismatched PNA, a 4-amino-acid (Gly-(D)-Ala-Gly-Gly) linker forming an N-4 structure was used for coupling Tc-99m. Probes were labeled with Tc-99m by ligand exchange. The radiochemical purity of these Tc-99m-labeled probes was determined by reversed-phase high-performance liquid chromatography. Cellular uptake, retention, binding specificity, and stability of the probes were studied either in vitro or in vivo. Biodistribution and radionuclide imaging were performed in BALB/c nude mice bearing SKOV3 (EGFR-positive) or MDA-MB-435S (EGFR-negative) carcinoma xenografts, respectively. Results: The average labeling efficiencies of 99mTc-EGFR-PNA and Tc-99m-CTL-PNA were 98.80% +/- 1.14% and 98.63% +/- 1.36% (mean +/- SD, n = 6), respectively, within 6 h at room temperature, and the radiochemical purity of the probes was higher than 95%. (99m)TcEGFR-PNA was highly stable in normal saline and fresh human serum at 37 degrees C in vitro and in urine and plasma samples of nude mice after 2-3 h of injection. Cellular uptake and retention ratios of Tc-99m-EGFR-PNA in SKOV3 cells were higher than those of Tc-99m-CTL-PNA and the EGFR-negative control. Meanwhile, EGFR mRNA binding (99m)TcEGFR-PNA was blocked with an excess of unlabeled EGFR-PNA in SKOV3 cell lines. The biodistribution study demonstrated accumulation of Tc-99m-EGFR-PNA primarily in the SKOV3 xenografts and in EGFR-expressing organs. Radionuclide imaging demonstrated clear localization of Tc-99m-EGFR-PNA in the SKOV3 xenografts shortly after injection but not in Tc-99m-CTL-PNA and the EGFR-negative control. Conclusion: Tc-99m-EGFR-PNA has the potential for imaging EGFR mRNA overexpression in tumors.