The gastrin-releasing peptide receptor (GRP-r) is over-expressed in various human tumors. Recently, 99mTc-EDDA/HYNIC-Lys3-bombesin (99mTc-BN) was reported as a radiopharmaceutical with specific cell GRP-r binding and images in breast cancer patients demonstrated distinct radioactivity accumulation in malignant tissue. The HIV Tat-derived peptide has been used to deliver a large variety of cargoes into cells. Therefore, a new hybrid radiopharmaceutical of type 99mTc-N2S2-Tat(49–57)-Lys3-bombesin (99mTc-Tat-BN) would increase cell uptake. The aim of this research was to prepare and assess in vitro and in vivo uptake kinetics in cancer cells of 99mTc-Tat-BN and to compare its cellular internalization with that of 99mTc-BN. Structures of N2S2-Tat-BN and Tc(O)N2S2-Tat-BN were calculated by an MM procedure. 99mTc-Tat-BN was synthesized and stability studies carried out by HPLC and ITLC-SG analyses in serum and cysteine solutions. In vitro internalization was tested using human prostate cancer PC-3 cells and breast carcinoma cell lines MDA-MB231 and MCF7. Biodistribution was determined in PC-3 tumor-bearing nude mice. Results showed a minimum energy of 271 kcal/mol for N2S2-Tat-BN and 300 kcal/mol for Tc(O)N2S2-Tat-BN. 99mTc-Tat-BN radiochemical purity was > 90%. In vitro studies demonstrated stability in serum and cysteine solutions, specific cell receptor binding and internalization in three cell lines was significantly higher than that of 99mTc-BN (p < 0.05). The tumor-to-muscle radioactivity ratio was 8.5 for 99mTc-Tat-BN and 7 for 99mTc-BN. Therefore, this hybrid is potentially useful in breast and prostate cancer imaging.