Over the last several decades, a number of experimental studies have demonstrated that a large transmural post-myocardial infarction (MI) scar, primarily in rats, contains areas with surviving cardiac myocytes which are remotely separated from the remaining non-infarcted myocardium of the left ventricular (LV) free wall. However, until now, a little attention was paid to the condition of such cardiac myocytes as well to the microenvironment in which they resided. Accordingly, our current investigation has been undertaken in order to thoroughly characterize these cells and their surroundings during development and maturation of the post-MI scar. A large transmural MI (greater than 50% of the LV free wall) was induced in 12-month-old male Sprague-Dawley rats by permanent ligation of the left coronary artery. The rats were euthanized 3 days, 1, 2, 4, 8 and 12 weeks after MI, and their hearts were collected for evaluation. The histological cross-sections taken from mid-ventricular level of the hearts were stained with various histological and immunofluorescence techniques. Digital imaging and quantitative image analysis were conducted using an Olympus BX53 fluorescence microscope equipped with a DP72 digital camera and Image-Pro Analyzer 7 software. We found that between day 3 and 12 weeks post-MI, the infarcted/scarred area of the LV free wall has retained the small isolated groups of two regionally distinct populations of surviving cardiac myocytes: 1) subendocardial and 2) subepicardial (perivenous). The analysis of fractional tissue volume content within the same-sized post-MI scars revealed that surviving cardiac myocytes occupied relatively similar volume after 4, 8 and 12 weeks (3.3±0.4%, 3.1±0.8% and 2.4±0.4%, respectively). However, while the mean cross-sectional area of subepicardial (perivenous) myocyte showed a significant increase between 1 and 12 weeks post-MI (251.7±22.6 μm2 vs. 325.4±8.5 μm2, P≤0.05), the mean area of subendocardial myocytes underwent a marked reduction during the same period (208.4±9.4 μm2 vs. 152.8±10.7 μm2, P≤0.01). A substantial atrophy of subendocardial myocytes was associated with a progressive accumulation of fibro-elastic extracellular matrix around these cells. At the same time, an apparent enlargement of perivenous (subepicardial) myocytes coincided with development of the well-defined microcirculatory network in the vicinity of such myocytes. Despite the opposite dynamics in the changes of the cell size, both populations of surviving cardiac myocytes displayed the presence of contractile proteins organized in sarcomeres, myocyte-to-myocyte coupling via the intercalated discs and gap junctions as well as existence of focal glycogen deposits in the cytoplasm. Therefore, our findings are the first to emphasize the fact that the post-MI scar of middle-aged rats has two regionally distinct populations of surviving cardiac myocytes, which can be considered for further investigation as a potential source of the endogenous contractile cells suitable for restoration of the functional myocardium in place of the scar tissue.