Acute myocardial infarction (MI), which involves the rupture of existing atheromatous plaque, remains highly unpredictable despite recent advances in the diagnosis and treatment of coronary artery disease. Accordingly, a clinical measurement that can predict an impending MI is desperately needed. Here, we characterize circulating endothelial cells (CECs) using an automated and clinically feasible CEC three-channel fluorescence microscopy assay in 50 consecutive patients with ST-segment elevation MI and 44 consecutive healthy controls. CEC counts were significantly elevated in MI cases versus controls, with median numbers of 19 and 4 cells/ml, respectively (P = 1.1 × 10(-10)). A receiver-operating characteristic (ROC) curve analysis demonstrated an area under the ROC curve of 0.95, suggesting near-dichotomization of MI cases versus controls. We observed no correlation between CECs and typical markers of myocardial necrosis (ρ = 0.02, creatine kinase-myocardial band; ρ = -0.03, troponin). Morphological analysis of the microscopy images of CECs revealed a 2.5-fold increase (P < 0.0001) in cellular area and a twofold increase (P < 0.0001) in nuclear area of MI CECs versus healthy controls, age-matched CECs, as well as CECs obtained from patients with preexisting peripheral vascular disease. The distribution of CEC images that contained from 2 to 10 nuclei demonstrates that MI patients were the only subject group to contain more than 3 nuclei per image, indicating that multicellular and multinuclear clusters are specific for acute MI. These data indicate that CEC counts may serve as a promising clinical measure for the prediction of atherosclerotic plaque rupture events.