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The aim of the present study was to prepare 99mTc-folate-glucaric acid and investigate the radiopharmaceutical potential for tumors imaging that over express folate receptor. Folate-glucaric acid was synthesized and the synthesized folate conjugate was confirmed with 1H NMR and LC–MS/MS methods. Folate-glucaric acid was labeled with 99mTc, and its radiolabelling efficiency was found as 96 ± 2.0%. Biodistribution study of 99mTc-folate-glucaric acid was carried out in vivo using two groups of female Albino Wistar rats: folate receptor (FR) saturated and unsaturated. Biodistribution study showed that 99mTc-folate-glucaric acid indicated high uptake in folate receptor rich tissues such as breast, ovary and uterus. Therefore, 99mTc-folate-glucaric acid shows good radiolabeling and biodistribution in FR organs, the radiolabeled conjugate is a reason potentially useful radiopharmaceutical for detection of FR-positive tumors.