We propose an enhanced method to accurately retrieve time-activity curves (TACs) of blood and tissue from dynamic 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography (PET) cardiac images of mice. The method is noninvasive and consists of using a constrained nonnegative matrix factorization algorithm (CNMF) applied to the matrix (A) containing the intensity values of the voxels of the left ventricle (LV) PET image. CNMF factorizesAinto nonnegative matricesHandW, respectively, representing the physiological factors (blood and tissue) and their associated weights, by minimizing an extended cost function. We verified our method on 32 C57BL/6 mice, 14 of them with acute myocardial infarction (AMI). With CNMF, we could break down the mouse LV into myocardial and blood pool images. Their corresponding TACs were used in kinetic modeling to readily determine the [18F]FDG influx constant (Ki) required to compute the myocardial metabolic rate of glucose. The calculatedKivalues using CNMF for the heart of control mice were in good agreement with those published in the literature. Significant differences inKivalues for the heart of control and AMI mice were found using CNMF. The values of the elements ofWagreed well with the LV structural changes induced by ligation of the left coronary artery. CNMF was compared with the recently published method based on robust unmixing of dynamic sequences using regions of interest (RUDUR). A clear improvement of signal separation was observed with CNMF compared to the RUDUR method.