Background: Although there have been reports on threadlike structures inside the heart, they have received little attention. We aimed to develop a method for observing such structures and to reveal their ultrastructures. Methods: An in situ staining method, which uses a series of procedures of 0.2–0.4% trypan blue spraying and washing, was applied to observe threadlike structures on the surfaces of endocardia. The threadlike structures were isolated and observed by using confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Results: Networks of endocardial vessels (20 µm in thickness) with expansions (40–100 µm in diameter) were visualized; they were movable on the endocardium of the bovine atrium and ventricle. CLSM showed that (1) rod-shaped nuclei were aligned along the longitudinal direction of the endocardial vessel and (2) there were many cells inside the expansion. TEM on the endocardial vessel revealed that (1) there existed multiple lumens (1–7 µm in diameter) and (2) the extracellular matrices mostly consisted of collagen fibers, which were aligned along the longitudinal direction of the endocardial vessel or were locally organized in reticular structures. Conclusion: We investigated the endocardial circulatory system in bovine cardiac chambers and its ultrastructures, such as nucleic distributions, microlumens, and collagenous extracellular matrices.