Technetium labeled isonitrile analogues are widely used as myocardial perfusion imaging agents. We synthesized and characterized a new isonitrile compound, ethyl 3- isocyanobutyrate (EIB). Proton and 13C NMR spectroscopy and thin layer chromatography with a C18 coat was performed. EIB was easily labeled with 99mTcO4-with sodium dithionite. The labeling efficiency measured by RP-HPLC was over 95%. The labeled product was stable with dilution in normal saline and with prolonged incubation at room temperature. There was no formation of secondary products or free 99mTcO4. In vivo kinetics study of Tc-99m(I) labeled EIB in rabbits showed adequate myocardial uptake, good contrast against lung background, and relatively rapid liver clearance. The heart to lung ratio was over 2.5 and the heart to liver ratio was approximately from 0.4 to 5 at 60 minutes post injection. Hepatic clearance of Tc-99m-MIBI was faster (t1/2,=6 minutes) than that of Tc-99m-MIBI. In vivo kinetics observed in dog was similar to that in rabbit but there was faster gallbladder filling, and thus lower liver background. SPECT imaging of the canine myocardium showed favorable imaging characteristics. However, biodistribution in mice demonstrated a myocardial % injected dose/organ of less than 0.1%. This was thought to be due to interspecies difference in plasma esterase activity. In human plasma, Tc-99m(I) labeled EIB was stable for at least 2 hours, without production of secondary products by HPLC. We conclude that ethyl 3-isocyanobutyrate may be a potential new myocardial perfusion imaging agent and deserves further investigation as to its usefulness for clinical use.