Cardiac P2X purinergic receptors can mediate an increase in myocyte contractility and a potentially important role in the heart. The P2X₄ receptor (P2X₄R) is an important subunit of native cardiac P2X receptors. Using transgenic mice with cardiac-specific overexpression of P2X₄R (Tg) as a model, the objectives here were to characterize the P2X receptor-mediated cellular contractile and Ca²⁺ transient effects and to determine the mechanism underlying the receptor-induced increase in myocyte contractility. In response to agonist 2-meSATP, Tg myocytes showed an increased intracellular Ca²⁺ transient, as defined by fura 2 fluorescence ratio, and an enhanced contraction shortening that were unaccompanied by cyclicAMP accumulation or L-type Ca²⁺ channel activation. The increased Ca²⁺ transient was not associated with any alteration in action potential duration, resting membrane potential, or diastolic fluorescence ratio or rates of rise and decline of the Ca²⁺ transient. Simultaneous Ca²⁺ transient and contraction measurements did not show any agonist-mediated change in myofilament Ca²⁺ sensitivity. However, activation of the overexpressed P2X4 receptor caused an enhanced SR Ca²⁺ loading, as evidenced by a 2-meSATP-evoked increase in the caffeine-induced inward current and Ca²⁺ transient. Similar data were obtained in wild type mouse ventricular myocytes. Thus, an increased SR Ca²⁺ content, occurring in the absence of cyclic AMP accumulation or L-type Ca²⁺ channel activation, is the principal mechanism by which cardiac P2X receptor mediates a stimulatory effect on cardiac myocyte contractility.