Agonist properties of the P2X(7) receptor (P2X(7)R) differ strikingly from other P2X receptors in two main ways: high concentrations of ATP (> 100 mu M) are required to activate the receptor, and the ATP analog 2', 3'-O-(4-benzoyl-benzoyl) ATP (BzATP) is both more potent than ATP and evokes a higher maximum current. However, there are striking species differences in these properties. We sought to exploit the large differences in ATP and BzATP responses between rat and mouse P2X(7)R to delineate regions or specific residues that may be responsible for the unique actions of these agonists at the P2X(7)R. We measured membrane currents in response to ATP and BzATP at wild-type rat and mouse P2X(7)R, at chimeric P2X(7)Rs, and at mouse P2X(7)Rs bearing point mutations. Wild-type rat P2X(7)R was 10 times more sensitive to ATP and 100 times more sensitive to BzATP than wild-type mouse P2X(7)R. We found that agonist EC50 values were determined solely by the ectodomain of the P2X(7)R. Two segments (residues 115-136 and 282-288), when transposed together, converted mouse sensitivities to those of rat. Point mutations through these regions revealed a single residue, asparagine(284), in the rat P2X(7)R that fully accounted for the 10-fold difference in ATP sensitivity, whereas the 100-fold difference in BzATP sensitivity required the transfer of both Lys(127) and Asn(284) from rat to mouse. Thus, single amino acid differences between species can account for large changes in agonist effectiveness and differentiate between the two widely used agonists at P2X(7) receptors.