Activation of cardiac P2X receptor can stimulate an increase in myocyte contractility. However, the underlying mechanism is not known. The objective here is to investigate the positive inotropic mechanism of cardiac P2X receptor using mice with cardiac-specific overexpression of the human P2X4 receptor (Tg). The P2X agonist 2-meSATP can evoke a greater inward current in Tg than in wild type (WT) cells. We tested the hypothesis that an increased P2X receptor-mediated inward current is associated with an increased sarcoplasmic reticulum (SR) Ca2+ loading and that this increased SR content is a mechanism for the enhanced myocyte contractility mediated via P2X receptors. The SR content was determined by the Na+/Ca++ exchanger-mediated inward current during a rapid caffeine pulse. Activation of the overexpressed P2X4 receptor by 2-meSATP caused a significant increase in the caffeine-mediated inward current, showing an augmented SR Ca2+ loading by the agonist in Tg cells. To determine whether P2X receptor activation alters myofilament Ca2+ sensitivity, simultaneous measurement of Ca2+ transients by fura-2 fluorescence ratio and cell shortening (CS) were determined in Tg cells at 0.2 to 2Hz stimulus frequencies. The plot of CS against fura-2 fluorescence ratio at different Hz was linear and similar before and after 2-meSATP, suggesting that P2X receptor activation does not change myofilament Ca2+sensitivity. 2-meSATP did not change L-type Ca current in Tg or WT cells and did not stimulate cyclic AMP accumulation. We conclude that activation of cardiac P2X receptor can augment SR Ca2+ content independent of cyclic AMP and that this increased content was associated with and likely contributed to the P2X receptor-mediated stimulation of myocyte contractility.