Background - The great clinical potential of myocardial β-AR imaging has been shown by recent studies evaluating the β-AR specific, non selective agent [11C]-CGP12177 in the setting of idiopathic dilated cardiomyopathy and myocardial infarction. However, the short half-life of 11C hampers the potential of [11C]-CGP12177 for routine clinical use. AMI9 is an analog of the β-adrenoceptor ligand practolol that can readily be labeled using radioactive isotopes of iodine. The present study was aimed at characterizing the in vitro, ex vivo, and in vivo β-AR binding properties of [125I]-AMI9. Methods and Results – Newborn rat cardiomyocytes were used for saturation and kinetic binding assays as well as for displacement and competition experiments. Isolated perfused rat hearts were used to evaluate the pharmacological activity of AMI9. The in vivo kinetics of [125I]-AMI9 were studied using biodistribution experiments in mice. [125I]-AMI9 displayed high specific affinity for β-AR with no β-AR subtype selectivity (KD, 5.6±0.3 nM; Bmax, 231±7 fmol/mg of protein). AMI9 potently inhibited the inotropic effects of isoproterenol. The early in vivo cardiac and lung activities of [125I]-AMI9 compared favorably with those of the clinically validated tracer CGP12177. Conclusions – Iodine-labeled AMI9 is a promising agent for the molecular imaging of myocardial β-AR density.