Introduction: In this study, I2P-RGD(2) was used as the example to illustrate a novel approach for dimerization of cyclic RGD peptides. The main objective of this study was to explore the impact of bifunctional linkers (glutamic acid vs. iminodiacetic acid) on tumor-targeting capability and excretion kinetics of the Tc-99m-labeled dimeric cyclic RGD peptides. Methods: HYNIC-I2P-RGD(2) was prepared by reacting I2P-RGD(2) with HYNIC-OSu in the presence of diisopropylethylamine, and was evaluated for its 04,133 binding affinity against I-125-echistatin bound to U87MG glioma cells. Tc-99m-I2P-RGD(2) was prepared with high specific activity (similar to 185 GBq/mu mol). The athymic nude mice bearing U87MG glioma xenografts were used to evaluate its biodistribution properties and image quality in comparison with those of Tc-99m-3P-RGD(2). Results: The IC50 value for HYNIC-I2P-RGD(2) was determined to be 39 +/- 6 nM, which was very close to that (IC50 = 33 +/- 5 nM) of HYNIC-3P-RGD(2). Replacing glutamic acid with iminodiacetic acid had little impact on a [33 binding affinity of cyclic RGD peptides. 99mTc-I2P-RGD(2) and 99mTc-3P-RGD(2) shared similar tumor uptake values over the 2 h period, and its a 133-specificity was demonstrated by a blocking experiment. The uptake of 99mTc-I2P-RGD2 was significantly lower than 99mTc-3P-RGD2 in the liver and kidneys. The U87MG glioma tumors were visualized by SPECT with excellent contrast using both Tc-99m-I2P-RGD(2) and Tc-99m-3P-RGD(2). Conclusion: Iminodiacetic acid is an excellent bifunctional linker for dimerization of cyclic RGD peptides. Bifunctional linkers have significant impact on the excretion kinetics of Tc-99m radiotracers. Because of its lower liver uptake and better tumor/liver ratios, Tc-99m-12P-RGD(2) may have advantages over Tc-99m-3P-RGD(2) for diagnosis of tumors in chest region. (C) 2017 Elsevier Inc. All rights reserved.