This study sought to evaluate K(HYNIC)(2) (K = lysine and HYNIC = 6-hydrazinonicotinyl) as a bifunctional chelator for (99m)Tc-labeling of biomolecule. In this study, four K(HYNIC)(2)-conjugated cyclic RGD peptides, K(HYNIC)(2)-RGD(2) (RGD(2) = E[c(RGDfK)](2)), K(HYNIC)(2)-3G-RGD(2) (3G-RGD(2) = Gly-Gly-Gly-E[Gly-Gly-Gly-c(RGDfK)](2)), K(HYNIC)(2)-2P-RGD(2) (2P-RGD(2) = E[PEG4-c(RGDfK)](2), and PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid), and K(HYNIC)(2)-3P-RGD(2) (3P-RGD(2) = PEG4-E[PEG4-c(RGDfK)]2) were prepared, and evaluated for their integrin αvβ3 binding affinity. IC(50) values were determined to be 47 ± 2, 35 ± 2, 37 ± 2, 85 ± 2, and 422 ± 15 nM for K(HYNIC)(2)-2P-RGD(2), K(HYNIC)(2)-3P-RGD(2), K(HYNIC)(2)-3G-RGD(2), K(HYNIC)(2)-RGD(2), and c(RGDyK), respectively, against (125)I-echistatin bound to U87MG cells. Macrocyclic complexes [(99m)Tc(K(HYNIC)(2)-RGD(2))(tricine)] (1), [(99m)Tc(K(HYNIC)(2)-3G-RGD(2))(tricine)] (2), [(99m)Tc(K(HYNIC)(2)-2P-RGD(2))(tricine)] (3), and [(99m)Tc(K(HYNIC)(2)-3P-RGD(2))(tricine)] (4) were prepared, and evaluated in athymic nude mice bearing U87MG glioma xenografts for their tumor targeting capability and biodistribution. It was found that 1-4 all had high solution stability and more than two isomers, as evidenced by the presence of multiple radiometric peaks in their radio-HPLC chromatograms. The tumor uptake of 1-4 was 3.78 ± 0.81, 7.46 ± 1.68, 9.74 ± 1.65, and 8.59 ± 1.52%ID/g, respectively, which was completely consistent with trend of integrin α(v)β(3) binding affinity for cyclic RGD peptides. Replacing [(99m)Tc(HYNIC)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3"-trisulfonate) with [(99m)Tc(K(HYNIC)(2))(tricine)] had little impact on radiotracer tumor uptake; but it had significant effect on the uptake of radiotracer in kidneys, lungs, and spleen. The tumor was clearly visualized by SPECT/CT with excellent contrast in a glioma-bearing mouse administered with 4. K(HYNIC)(2) would be particularly useful for (99m)Tc-labeling of small biomolecules with one or more disulfide linkages.