BACKGROUND 99mTc(CO)3-nitrilotriacetic acid, 99mTc(CO)3(NTA), is a new renal tubular agent with pharmacokinetic properties comparable to those of 131I-OIH but the clearance of 99mTc(CO)3(NTA) and 131I-OIH is still less than the clearance of PAH, the gold standard for the measurement of effective renal plasma flow. At physiological pH, dianionic 99mTc(CO)3(NTA) has a mononegative inner metal-coordination sphere and a mononegative uncoordinated carboxyl group. To evaluate alternate synthetic approaches, we assessed the importance of an uncoordinated carboxyl group, long considered essential for tubular transport, by evaluating the pharmacokinetics of three analogs with the 99mTc(CO)3(NTA) metal-coordination sphere but with uncharged pendant groups. METHODS 99mTc(CO)3 complexes with N-(2-acetamido)iminodiacetic acid (ADA), N-(2-hydroxyethyl)iminodiacetic acid (HDA) and N-(fluoroethyl)iminodiacetic acid (FEDA) were prepared using a tricarbonyl kit and isolated by HPLC. The pharmacokinetics were evaluated in Sprague-Dawley rats, with 131I-OIH as an internal control; urine was analyzed for metabolites. Plasma protein binding and erythrocyte uptake were determined from the 10min blood samples. Re(CO)3(FEDA), the analog of 99mTc(CO)3(FEDA), was prepared and characterized. RESULTS 99mTc(CO)3(ADA), 99mTc(CO)3(HDA) and 99mTc(CO)3(FEDA) were efficiently prepared as a single species with high radiochemical purities (>99%). These new monoanionic 99mTc(CO)3 tracers with uncharged dangling groups all showed rapid blood clearance and high specificity for renal excretion. Activity in the urine, as a percent of 131I-OIH at 10 and 60min, was 96% and 99% for ADA, 96% and 100% for HDA, and 100% and 99% for FEDA, respectively. Each new tracer was excreted unchanged in the urine. The Re(CO)3(FEDA) structure adds compelling evidence that such 99mTc(CO)3(NTA) analogs have metal-coordination spheres identical to that of 99mTc(CO)3(NTA). CONCLUSIONS New tracers lacking the negatively charged pendant carboxyl group previously thought to be essential for rapid renal extraction, 99mTc(CO)3(ADA), 99mTc(CO)3(HDA) and 99mTc(CO)3(FEDA), exhibit pharmacokinetics in rats comparable to those of 99mTc(CO)3(NTA) and 131I-OIH. Furthermore, these encouraging results in rats warrant evaluation of this new tracer type in humans.