Although P2X receptors are suggested to play a role in synaptic neurotransmission, the specific physiological role of each P2X receptor subtype remains largely unknown. We used cultured chick embryo ventricular myocytes as a model to study a potential physiological role of the P2X4 receptor in mediating the positive inotropic effect of ATP. The chick P2X4 receptor (cP2X4R) mRNA was expressed in the heart and the pharmacological features of the ATP-induced positive inotropic response were similar to those of the cP2X4R in terms of insensitivity to blockade by known P2 receptor antagonists and the ineffectiveness of adenosine 5′-(α,β-methylene)triphosphate as an agonist. Treatment of myocytes with antisense oligonucleotides specific to the 5′ region of cP2X4R abrogated the P2 agonist-stimulated 45Ca influx. Similarly, antisense oligonucleotide treatment also blocked the 2-methylthio-ATP-stimulated increase in contractile amplitude. The data suggest that the native P2X4 receptor is involved in mediating the P2 agonist-stimulated response in the heart. In characterizing the biochemical property of the P2X4 receptor, antibody against cP2X4R detected a 44-kDa and a 58-kDa protein in the immunoblot. Inhibition of N-linked glycosylation by tunicamycin converted the 58-kDa protein to the 44-kDa protein, suggesting that the 58-kDa protein was a glycosylated P2X4 receptor. The nonglycosylated 44-kDa P2X4 receptor was resistant to various detergent/aqueous extraction, consistent with a role of glycosylation in maintaining its detergent solubility and hydrophilicity. Cross-linking the cell surface proteins with N-hydroxysuccinimide-SS-biotin followed by affinity precipitation with streptavidin-conjugated agarose and subsequent immunoblotting with anti-cP2X4R showed that only the glycosylated 58-kDa P2X4 receptor was expressed on the cell surface, indicating an important role of glycosylation for the receptor's localization on the plasma membrane. These data revealed a novel physiologic function of the P2X4 receptor and suggested the importance of N-linked glycosylation in its cell surface expression and detergent solubility.