Due to the limited results achieved in the clinical treatment of heart failure, a new inotropic strategy of myosin motor activation has been developed. The lead molecule of myosin activator agents is omecamtiv mecarbil, which binds directly to the heavy chain of the cardiac β-myosin and enhances cardiac contractility by lengthening the lifetime of the acto-myosin complex and increasing the number of the active force-generating cross-bridges. In the absence of relevant data, the effect of omecamtiv mecarbil on canine cardiac ryanodine receptors (RyR 2) has been investigated in the present study by measuring the electrical activity of single RyR 2 channels incorporated into planar lipid bilayer. When applying 100 nM Ca2+ concentration on the cis side ([Ca2+]cis) omecamtiv mecarbil (1–10 μM) significantly increased the open probability and opening frequency of RyR 2, while the mean closed time was reduced. Mean open time was increased moderately by 10 μM omecamtiv mecarbil. When [Ca2+]cis was elevated to 322 and 735 nM, the effect of omecamtiv mecarbil on open probability was evident only at higher (3–10 μM) concentrations. All effects of omecamtiv mecarbil were fully reversible upon washout. Omecamtiv mecarbil (up to 10 μM) had no effect on the open probability of RyR 1, isolated from either canine or rabbit skeletal muscles. It is concluded that the direct stimulatory action of omecamtiv mecarbil on RyR 2 has to be taken into account when discussing the mechanism of action or the potential side effects of the compound.