During the past decade radiolabeled RGD-peptides have been extensively studied to develop site-directed targeting vectors for integrins. Integrins are heterodimeric cell-surface adhesion receptors, which are upregulated in cancer cells and neovasculature during tumor angiogenesis and recognize the RGD aminoacid sequence. In the present study, we report the synthesis and development of two derivatives of the Nε-Lys derivatized cyclic Arg-Gly-Asp-d-Phe-Lys peptide, namely of cRGDfKHis and cRGDfK-CPA (CPA: 3-l-Cysteine Propionic Acid), radiolabeled via the [99mTc(H2O)3(CO)3]+ metal aquaion at a high yield even at low concentrations of 10–5 M (>87%) for cRGDfK-10–5 M (>93%) for cRGDfK-CPA. Radiolabeled peptides were characterized with regard to their stability in saline, in His/Cys solutions, as well as in plasma, serum and tissue homogenates and were found to be practically stable. Internalization and efflux assays using αvβ3-receptor-positive MDA-MB 435 breast cancer cells showed a good percentage of quick internalization (29.1 ± 9.8% for 99mTc-HiscRGDfK and 37.0 ± 0.7% for 99mTc-CPA-cRGDfK at 15 min) and no retention of radioactivity for both derivatives. Their in vivo behavior was assessed in normal mice and pathological SCID mice bearing MDA-MB 435 ανβ3 positive breast tumors. Both presented fast blood clearance and elimination via both the urinary and hepatobiliary systems, with 99mTc-His-cRGDfK remaining for a longer time than 99mTc-CPA-cRGDfK in all organs examined. Tumor uptake 30 min pi was higher for 99mTc-CPAcRGDfK (4.2 ± 1.5% ID/g) than for 99mTc-His-cRGDfK (2.8 ± 1.5% ID/g). Dynamic scintigraphic studies showed that the tumor could be visualized better between 15 and 45 min pi for both radiolabeled compounds but low delineation occurred due to high abdominal background. It was finally noticed that the accumulated activity on the tumor site was depended on the size of the experimental tumor; the smaller the size, the higher was the radioactivity concentration.