The first human studies of a characterized radiopharmaceutical containing a { 99m Tc(CO) 3 } + core, Na[ 99m Tc(CO) 3 (LAN)], demonstrated that Na[ 99m Tc(CO) 3 (LAN)] was an excellent renal imaging agent; however, its clearance was less than that of 131 I-orthoiodohippurate ( 131 I-OIH), and it did not provide a direct measure of effective renal plasma flow. In order to develop a 99m Tc renal agent with pharmacokinetic properties equivalent to those of 131 I-OIH, we investigated the 99m Tc(CO) 3 /Re(CO) 3 complexes formed from carboxymethylmercaptosuccinic acid (CMSAH 3 ) and thiodisuccinic acid (TDSAH 4 ). Once the ligand is bound to 99m Tc(CO) 3 through a thioether and two carboxyl groups, the complexes have at least one unbound carboxyl group, essential for the interaction with the renal tubular transporter.X-ray crystal structural analysis of [NMe 4 ][Re(CO) 3 (CMSAH)] was performed to interpret the nature of 99m Tc tracers. CMSAH 3 and TDSAH 4 were radiolabeled by incubating each ligand and the precursor [ 99m Tc(CO) 3 (H 2 O) 3 ] + at 70°C (pH 7) for 30 min. The products were purified by reversed-phase high-performance liquid chromatography, and biodistribution studies were performed in Sprague–Dawley rats, with 131 I-OIH as an internal control at 10 and 60 min.Radiolabeling CMSAH 3 and TDSAH 4 with the [ 99m Tc(CO) 3 (H 2 O) 3 ] + precursor gave products quantitatively. Analysis of the Re(CO) 3 complexes with the CMSAH 3 and TDSAH 4 ligands demonstrates that ligands are bound in 99m Tc/Re(CO) 3 complexes through a thioether and two deprotonated carboxyl groups (forming tridentate dianionic moieties, generally with two 5-membered chelate rings). Renal excretion at 60 min (activity in the urine as a percentage of 131 I-OIH) was 68±1% for Na 3 [ 99m Tc(CO) 3 (TDSA)] but was 98±1% for Na 2 [ 99m Tc(CO) 3 (CMSA)].In rats, Na 2 [ 99m Tc(CO) 3 (CMSA)] is extracted by the kidneys and eliminated in the urine almost as rapidly as 131 I-OIH; consequently, Na 2 [ 99m Tc(CO) 3 (CMSA)] may provide a direct measure of effective renal plasma flow, and further evaluation in humans is warranted.