We have shown previously that the epidermal growth factor peptide (EGF) may be radiolabeled with 99mTc at room temperature and neutral pH by using the N-hydroxysuccinimide ester of S-acetyl mercaptoacetyltriglycine (MAG3) as a bifunctional chelator. By a competition binding assay, we found that MAG3-conjugated EGF retained biological activity. Furthermore, the labeled peptide exhibited saturation binding to EGF receptor-positive tumor cell lines which could be inhibited by presaturation of the cells with unlabeled, native EGF. Biodistribution in normal mice at 3 h postadministration showed rapid clearance with minimal retention of the label in sampled organs. We have now investigated the tumor localization properties in mice of this labeled peptide. Nude mice implanted with the EGF receptor-positive tumors A431 and LS-174T were administered labeled EGF and a labeled control peptide (BPTI, aprotinin). Tumor uptake at 12 h postadministration was 0.44% injected dose/g for EGF/g vs. 0.09 for the control. Pretreatment of tumored mice with unlabeled EGF blocked about half the tumor uptake. Animals were also administered an anti-EGF receptor antibody labeled with 99mTc via MAG3. Relative to the antibody, tumor-to-muscle ratios were improved from 6 to 15 and tumor-to-blood ratios from 0.4 to 7 with EGF. These favorable results along with documented evidence of overexpression of the EGF receptor in many human tumors suggest that 99mTc-EGF should be considered further for tumor detection.