Ca2+ mobilization via inositol triphosphate receptors (IP3Rs) results in cytosolic Ca2+ elevation in cardiac stem cells, raising the possibility that IP3R channels are preserved and functional in differentiated myocytes. Thus, LV myocytes were obtained from human and mouse hearts and the expression and function of IP3Rs were established. By immunocytochemistry, IP3Rs were predominantly distributed in the perinuclear region and minimally in the sarcomeres. In field stimulated cells, IP3R activation via agonists of Gq-protein receptors (ET-1, ATP) or enhanced IP3R-IP3 affinity (thimerosal), increased diastolic Ca2+, Ca2+ transient amplitude and sarcomere shortening by 11%, 120% and 190%, respectively. Additionally, the release of extra-systolic Ca2+ occurred during the decay phase, leading to after-contractions. These effects were prevented by inhibition of IP3 production or IP3R blockade. In patch-clamped myocytes, changes in Ca2+ transient properties following IP3R activation were accompanied by a decrease in resting potential (4.9±0.5 mV), action potential (AP) prolongation and early afterdepolarization (EADs). Since these alterations may result from Ca2+ mobilization mediated by stimulation of IP3Rs or from enhanced RyR activity, voltage-clamp experiments were performed in Fluo-3 loaded cells. The ability of Ca2+ current to trigger the release of Ca2+ from RyRs was preserved after activation of IP3Rs. Moreover, Ca2+ transients evoked by depolarizing steps did not change in amplitude or display extra-systolic release. The net inward holding current was increased, consistent with enhanced Ca2+ extrusion via Na-Ca exchanger. In AP-voltage-clamp mode, prolonged APs with EADs led to increased Ca2+ transients and extra-systolic Ca2+ releases, mimicking IP3R activation. In contrast, in the presence of IP3R stimulation, normal-APs restored Ca2+ transients. In conclusion, Ca2+ mobilization via IP3Rs directly affects the electrical activity of human and rodent myocytes resulting in altered RyR-Ca2+ release and contractility. Ca2+ mobilization via inositol triphosphate receptors (IP3Rs) results in cytosolic Ca2+ elevation in cardiac stem cells, raising the possibility that IP3R channels are preserved and functional in differentiated myocytes. Thus, LV myocytes were obtained from human and mouse hearts and the expression and function of IP3Rs were established. By immunocytochemistry, IP3Rs were predominantly distributed in the perinuclear region and minimally in the sarcomeres. In field stimulated cells, IP3R activation via agonists of Gq-protein receptors (ET-1, ATP) or enhanced IP3R-IP3 affinity (thimerosal), increased diastolic Ca2+, Ca2+ transient amplitude and sarcomere shortening by 11%, 120% and 190%, respectively. Additionally, the release of extra-systolic Ca2+ occurred during the decay phase, leading to after-contractions. These effects were prevented by inhibition of IP3 production or IP3R blockade. In patch-clamped myocytes, changes in Ca2+ transient properties following IP3R activation were accompanied by a decrease in resting potential (4.9±0.5 mV), action potential (AP) prolongation and early afterdepolarization (EADs). Since these alterations may result from Ca2+ mobilization mediated by stimulation of IP3Rs or from enhanced RyR activity, voltage-clamp experiments were performed in Fluo-3 loaded cells. The ability of Ca2+ current to trigger the release of Ca2+ from RyRs was preserved after activation of IP3Rs. Moreover, Ca2+ transients evoked by depolarizing steps did not change in amplitude or display extra-systolic release. The net inward holding current was increased, consistent with enhanced Ca2+ extrusion via Na-Ca exchanger. In AP-voltage-clamp mode, prolonged APs with EADs led to increased Ca2+ transients and extra-systolic Ca2+ releases, mimicking IP3R activation. In contrast, in the presence of IP3R stimulation, normal-APs restored Ca2+ transients. In conclusion, Ca2+ mobilization via IP3Rs directly affects the electrical activity of human and rodent myocytes resulting in altered RyR-Ca2+ release and contractility.