The P2X4 receptor is involved in endothelium-dependent changes in large arterial tone in response to shear stress and is, therefore, potentially relevant to arterial compliance and pulse pressure. Four identified nonsynonymous polymorphisms in P2RX4 were reproduced in recombinantly expressed human P2X4. Electrophysiological studies showed that one of these, the Tyr315>Cys mutation (rs28360472), significantly reduced the peak amplitude of the ATP-induced inward current to 10.9% of wild-type P2X4 receptors in transfected HEK-293 cells (10 μmol/L of ATP; n=4–8 cells; P<0.001). Concentration-response curves for ATP and the partial agonist BzATP demonstrate that the 315Cys-P2X4 mutant had an increased EC50 value for both ligands. Mutation of Tyr315>Cys likely disrupts the agonist binding site of P2X4 receptors, a finding supported by molecular modeling based on the zebrafish P2X4 receptor crystal structure. We tested inheritance of rs28360472 encoding the Tyr315>Cys mutation in P2RX4 against pulse pressure in 2874 subjects from the Victorian Family Heart Study. The minor allele frequency was 0.014 (1.4%). In a variance components analysis we found significant association with pulse pressure (P=0.023 for total association) where 1 minor allele increased pulse pressure by 2.84 mm Hg (95% CI: 0.41–5.27). This increase in pulse pressure associated with inheritance of 315Cys-P2X4 receptors might reflect reduced large arterial compliance as a result of impaired endothelium-dependent vasodilation in large arteries.