This report describes the evaluation of three cationic 99m Tc(I)–tricarbonyl complexes — [ 99m Tc(CO) 3 (L)] + (L=N-methoxyethyl-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (ME-PNP), N-[15-crown-5)-2-yl]-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (15C5-PNP) and N-[18-crown-6)-2-yl]-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (18C6-PNP)) — as potential radiotracers for myocardial perfusion imaging. Biodistribution, imaging and metabolism studies were performed using Sprague–Dawley rats. It was found that bisphosphine ligands have a significant impact on the biodistribution characteristics and clearance kinetics of their cationic 99m Tc(I)–tricarbonyl complexes. Among the three radiotracers evaluated in this study, [ 99m Tc(CO) 3 (15C5-PNP)] + has a very high initial heart uptake and is retained in the rat myocardium for >2 h. It also shows rapid clearance from the liver and lungs. The heart/liver ratio of [ 99m Tc(CO) 3 (15C5-PNP)] + is ∼2.5 times better than that of 99m Tc-sestamibi at 30 min postinjection. [ 99m Tc(CO) 3 (15C5-PNP)] + is almost identical to 99m TcN-DBODC5 with respect to heart uptake, heart/lung ratio and heart/liver ratio. Results from metabolism studies show that there is no significant metabolism for [ 99m Tc(CO) 3 (15C5-PNP)] + in the urine, but it does show a small metabolite peak (<10%) in the radio high-performance liquid chromatography chromatogram of the feces sample at 120 min postinjection. Results planar imaging studies demonstrate that [ 99m Tc(CO) 3 (15C5-PNP)] + has a much better liver clearance profile than 99m Tc-sestamibi and might give clinically useful images of the heart as early as 30 min postinjection. [ 99m Tc(CO) 3 (15C5-PNP)] + is a very promising candidate for more preclinical evaluations in various animal models.