We have previously demonstrated the existence of an adenosine 5',5"',P1,P4-tetraphosphate (Ap4A) receptor in mouse hart membrane fractions [Hilderman, R. H., Martin, M., Zimmerman, J. K., & Pivorun, E. P. (1991) J. Biol. Chem. 266, 6915-6918]. However, we did not determine the cellular localization or distribution of the receptor. In this report, the Ap4A receptor is shown to be on the cell surface of individual mouse heart cells by the following four methods: (1) intact cells show specific, saturable, and reversible binding of Ap4A; (2) monoclonal antibodies (Mabs) raised against the Ap4A receptor inhibit Ap4A binding to its receptor on intact heart cells; (3) bound Mabs are shown to be at the outer cell surface via reaction with a alkaline phosphatase conjugated goat anti-rat IgG; (4) when intact cells are labeled with the impermeable cell surface labeling reagent, (sulfosuccinimido) biotin, labeled receptor is immunoprecipitated with Mabs. Furthermore, subcellular fractionation of mouse hearts demonstrates that virtually all of the Ap4A receptor is associated with a membrane fraction with at least 77% of the active receptor on plasma membranes.