Inositol 1,4,5-trisphosphate receptor (IP₃R)-dependent Ca²⁺ signaling exerts positive inotropic, but also arrhythmogenic effects on excitation-contraction coupling (ECC) in the atrial myocardium. The role of IP₃R-dependent sarcoplasmic reticulum (SR) Ca²⁺ release for ECC in the ventricular myocardium remains controversial. Here, we investigated the role of this signaling pathway during ECC in isolated rabbit ventricular myocytes. Immunoblotting of proteins from ventricular myocytes showed expression of both Type 2 and Type 3 IP₃R at levels ~3.5 fold less than in atrial myocytes. In permeabilized myocytes, direct application of IP₃ (10 µM) produced a transient 21% increase in the frequency of Ca²⁺ sparks (P<0.05). This increase was accompanied by a 13% decrease in spark amplitude (P<0.05) and a 7% decrease in SR Ca²⁺ load (P<0.05), and was inhibited by IP₃R antagonists 2-APB (20 µM) and heparin (0.5 mg/mL). In intact myocytes endothelin-1 (100 nM) was used to stimulate IP₃ production, and caused a 38% (P<0.05) increase in the amplitude of action potential-induced (0.5 Hz, field stimulation) Ca²⁺ transients. This effect was abolished by the IP₃R antagonist 2-APB (2 µM) or using adenoviral expression of an IP₃ affinity trap that buffers cellular IP₃. Taken together, these data suggest that in rabbit ventricular myocytes IP₃R-dependent Ca²⁺ release has positive inotropic effects on ECC by facilitating Ca²⁺ release through RyR clusters.