ATP-gated P2X 4 purinergic receptors (P2X 4 Rs) are cation channels with important roles in diverse cell types. To date, lack of specific inhibitors has hampered investigations on P2X 4 Rs. Recently, the benzodiazepine derivative, 5-BDBD has been proposed to selectively inhibit P2X 4 Rs. However, limited evidences are currently available on its inhibitory properties. Thus, we aimed to characterize the inhibitory effects of 5-BDBD on recombinant human P2X 4 Rs. We investigated ATP-induced intracellular Ca 2+ signals and whole cell ion currents in HEK 293 cells that were either transiently or stably transfected with hP2X 4 Rs. Our data show that ATP (< 1 μM) stimulates P2X 4 R-mediated Ca 2+ influx while endogenously expressed P2Y receptors are not activated to any significant extent. Both 5-BDBD and TNP-ATP inhibit ATP-induced Ca 2+ signals and inward ion currents in a concentration-dependent manner. Application of two different concentrations of 5-BDBD causes a rightward shift in ATP dose-response curve. Since the magnitude of maximal stimulation does not change, these data suggest that 5-BDBD may competitively inhibit the P2X 4 Rs. Our results demonstrate that application of submicromolar ATP concentrations allows reliable assessment of recombinant P2XR functions in HEK 293 cells. Furthermore, 5-BDBD and TNP-ATP have similar inhibitory potencies on the P2X 4 Rs although their mechanisms of actions are different.