ATP induces Ca²⁺ influx across the cell membrane and activates release from intracellular Ca²⁺ pools in vascular endothelial cells (ECs). Ca²⁺ signaling leads to the modification of a variety of EC functions, including the production of vasoactive substances such as nitric oxide and prostacyclin. However, the molecular mechanisms for ATP-induced Ca²⁺ influx in ECs have not been thoroughly clarified. Here we demonstrate evidence that a P2X₄receptor for an ATP-gated cation channel is predominantly expressed in human ECs and is involved in the ATP-induced Ca²⁺ influx. Northern blot analysis distinctly showed the expression of P2X₄ mRNA in human ECs cultured from the umbilical vein, aorta, pulmonary artery, and skin microvessels. Competitive PCR revealed that P2X₄ mRNA expression was much higher in ECs than was the expression of other subtypes, including P2X₁, P2X₃, P2X₅, and P2X₇. Treatment of ECs with antisense oligonucleotides designed to target the P2X₄ receptor decreased the P2X₄ mRNA and protein levels to ∼25% of control levels and markedly prevented the ATP-induced Ca²⁺ influx.