Atrial myocytes are subjected to shear stress under physiological and pathological conditions during cardiac cycle. It has been recently suggested that ATP release of atrial cells induces longitudinal Ca2+ wave via activation of P2Y1 purinergic signaling in rat atrial myocytes under shear stress. In the present study, we examined whether P2X purinoceptor, a non-selective cation channel, is activated in these cells using a whole-cell patch clamp combined with a pressurized microflow system. Shear stress of ~16 dyne/cm2(1-s long) produced a slow inward current(ISS) at −70 mV in symmetrical CsClrich solutions. Removal of external Ca2+ significantly enhanced the ISS by approximately 140%. Pre-treatment of suramin(10 µM), the general P2-purinoceptor antagonist, suppressed the ISS by ~70%. In addition, the ISS was inhibited by ~50% by pre-exposure to P2X purinoceptor inhibitor, iso-PPADS(100 µM). Inhibition of transient receptor potential melastatin 4, the Ca2+-activated non-selective cation channel, using 9-phenanthrol(30 µM) decreased ISS by ~30%. When the delayed rectifying K+ channels, that have slight permeability to Cs+, were blocked using 4-aminopyridine(200 µM), ISS was not altered. Our data suggest that shear stress activates P2X purinoceptors in atrial myocytes, thereby partly contributing to shear-activated slow cation influx at resting membrane potential.