Introduction: TMTP1 (NVVRQ) is a novel tumor-homing peptide, which specifically targets tumor metastases, even at the early stage of occult metastasis foci. Fusing TMTP1 to therapeutic peptides or proteins can increase its anti-cancer efficacy both in vivo and in vitro. Here, we labeled TMTP1 with Tc-99m to evaluate its targeting properties in an ovarian cancer xenograft tumor mouse model and a gastric cancer xenograft mouse model. Methods: The invasion ability of SKOV3 and highly metastatic SKOV3.ip cell lines were performed by the Transwell Invasion Assays, and then Rhodamine-TMTP1 was used to detect its affinity to these two cells. Using the co-ligand ethylenediamine-N, N'-diacetic acid (EDDA) and the bifunctional chelator 6-hydrazinonicotinic acid (HYNIC), the TMTP1 peptide was labeled with Tc-99m. A cell-binding assay was performed by incubating cancer cells with Tc-99m-HYNIC-TMTP1 with or without an excess dose of cold HYNIC-TMTP1. To evaluate the probe in vivo, nude mice bearing SKOV3, SKOV3.ip and MNK-45 tumor cells were established and subjected to SPECT imaging after injection with Tc-99m-HYNIC-TMTP1. Ex vivo gamma-counting of dissected tissues from the mice was used to evaluate its biodistribution. Results: Tc-99m-HYNIC-TMTP1 was successfully synthesized. The radiotracer also exhibited high hydrophilicity and excellent stability in vitro and in vivo. It has strong affinity to highly metastatic cancer cell lines but not to poorly metastatic cell lines. After mice were injected with Tc-99m-HYNIC-TMTP1, non-invasive SPECT imaging detected SKOV3.ip and MNK-45 xenograft tumors but not SKOV3 xenograft tumors. This result can be inhibited by excess HYNIC-TMTP1. The uptake of Tc-99m-HYNIC-TMTP1 in SKOV3.ip xenograft tumors was 0.182 +/- 0.017% ID/g at 2 h p.i. with high renal uptake (74.32 +/- 15.05% ID/g at 2 h p.i.). Conclusion: Tc-99m-HYNIC-TMTP1 biodistribution and SPECT imaging demonstrated its ability to target highly metastatic tumors. Therefore, metastasis can be non-invasively investigated by SPECT imaging using Tc-99m-HYNIC-TMTP1. Meanwhile, this radiotracer has some shortages in the low % ID/g of tumors and high accumulation in the kidney. (C) 2014 Elsevier Inc. All rights reserved.