Mixed ligand fac-tricarbonyl complex of [99mTc(CO)3-DMSA-MIBI] has been prepared starting from the precursor [99mTc(OH2)3(CO)3]+. The complex can be obtained in good yield and purity in a two-step procedure by first attaching meso-2,3-dimercaptosuccinic acid (DMSA, HOOCCH(SH)CH(SH)COOH) with [99mTc(OH2)3(CO)3]+, followed by addition of MIBI [tetrakis-2-methoxyisobutylisonitrile (CH3OC(CH3)2CH2-N≡C) copper(I) tetrafluoroborate] solution. The complex was characterized by TLC and HPLC and was studied by means of octanol-water partition coefficient, electrophoresis, stability in vitro, and normal mice experiment. Biodistribution in mice demonstrated that the complex showed higher myocardial uptake after 0.5-hour p.i. The ratios of heart/liver (%ID/g) in the case of 99mTc(CO)3-DMSA-MIBI was higher (1.88) than that observed in case of 99mTc-MIBI1 (0.93) after 0.5-hour p.i. (P<0.05). Results showed that the complex may be developed to a novel myocardial perfusion-imaging agent.