In this review we describe the history of almost one century lasting investigations which eventually provided evidence convincing that cardiac myocytes possess all elements of the system of synthesis, intracellular transport and release of acetylcholine (ACh) independent of parasympathetic cholinergic innervation. The myocytes synthesis and release of ACh is tightly connected with their contractile activity. Moreover, it is necessary for maintaining the balance of autonomic control of the heart, particularly important in the heart failure. It has an antyhypertrophic activity and protects myocardium against ischemic/reperfusion injury which shows that it is involved in the regulation of the important cellular signaling pathways. These properties of the non-neuronal ACh system of the heart also rise the hope that it might be used for the therapeutic measures.