Myocardial cell death is implicit in heart disease and a major underlying cause of heart failure (1). Despite rapid advances in diagnostic technology there is still no reliable method for directly quantifying cardiac cell death in vivo (2). Here we report a novel isatin-derived 125I-FITI SPECT radiotracer selective for the caspase-3 activity that underlies cell death. Using ICMT-11 as a scaffold, 125I-FITI was synthesised using a modified one-pot radiochemical reaction (3) with a bathophenanthroline catalyst (4). Myocardial infarction (40 min ischemia followed by reperfusion) was induced in 16 male C57Bl6 mice with 8 control mice undergoing sham surgery. Eighteen hours after surgery, cardiac function and viability were assessed in vivo using high frequency ultrasound and late gadolinium enhanced cardiac MRI (5). At 24 hours all animals underwent tail vein infusion of 0.5MBq of 125I-FITI or a non-functional 125I-FITI derivative. Animals were sacrificed after 15, 30, 60 and 120 minutes. Organs were collected and gamma counted for bio-distribution of tracer. Hearts immediately underwent sectioning, staining with cell viability dye TTC, followed by plating for autoradiography for high resolution ex vivo assessment of localisation of FITI within the infarcted hearts. Ex vivo bio-distribution studies showed FITI uptake was highest in the stomach, thyroid, salivary glands and small intestine. Uptake of tracer within the hearts of control animals was low at all time points and reduced further by one and two hours after infusion. Ultrasound and MRI showed that all infarcted hearts had impaired contraction and small to medium infarct sizes of 17-38% (FIG). TTC staining concurred with this result (FIG). Autoradiographic assessment of radiotracer distribution within tissues showed high FITI accumulation within anterolateral regions of the myocardium (FigC). Comparison of FITI distribution with the in vivo MRI and in vitro TTC staining performed on the same hearts confirmed that these regions of tracer uptake corresponded with the location of the infarct. Importantly, the non-functional FITI derivative did not accumulate within the infarct region, indicating specificity of FITI binding. Sensitivity and specificity analysis confirmed accumulation of FITI within infarcted segments of the myocardium and that non-functional FITI had much lower sensitivity for myocardial cell death than the active compound. These data suggest that FITI accumulation is greatest at the site where cell death is maximal at 24 hours after myocardial infarction and that FITI is specifically binding the activated caspase3 present acuity after myocardial infarction. FITI may offer a novel imaging biomarker for quantification of cardiac cell death in vivo.