P2X₇ is an ATP-gated cation channel expressed primarily in cells of the immune system, where brief activation couples to pro-inflammatory cytokine release and prolonged activation leads to cell death. Uniquely for P2X₇, the agonist 2’, 3’-(benzoyl-4-benzoyl) ATP (BzATP) is more potent than ATP; in addition, striking differences in ATP and BzATP potency are observed between receptors from different species. To address the basis of differential agonist sensitivity, we firstly compared the ATP- and BzATP-induced currents of wild-type rat and mouse P2X₇ expressed in HEK-293 cells. Mouse P2X₇ displayed an 8-fold lower sensitivity to ATP (EC₅₀ values of 936±21μM compared to 123±4μM) and an 80-fold lower sensitivity to BzATP than rat P2X₇ (EC₅₀ values of 285±16μM compared to 3.6±0.2μM). Secondly, we analysed the agonist sensitivities of rat/mouse receptor chimaeras. We found that transposition of two segments of the ectodomain of rat P2X₇ (115-136 and 282-288) into mouse P2X₇ converted mouse P2X₇ agonist sensitivities to those of rat P2X₇. Finally, single point mutagenesis within these regions revealed two critical amino acids involved in agonist sensitivity. Substitution of asparagine-284 of the rat receptor into mouse P2X₇ (D284N) restored the level of ATP sensitivity to that of rat P2X₇ (EC₅₀ of 146±11μM). We demonstrated that this mutation increased the apparent molecular mass of the receptor from that of mouse P2X₇ (75kDa) to that of rat P2X₇ (78kDa), due to the incorporation of an additional N-glycosylation acceptor sequence (already present in rat P2X₇). We suggest that N-glycosylation at this position stabilises the structure of the receptor, and effects an increase in ATP potency through more efficient channel gating. Substitution of lysine-127 into mouse P2X₇ (A127K) enhanced BzATP but not ATP sensitivity (EC₅₀ values of 80±7μM and 815±56μM), suggesting a direct role for this residue in BzATP binding. A mouse P2X₇ construct bearing both mutations (A127K/D284N) was indistinguishable from rat P2X₇ in terms of ATP and BzATP sensitivity (EC₅₀ values of 112±11μM and 5±1μM respectively), demonstrating that lysine-127 and asparagine-284 are sufficient to confer rat-like ATP and BzATP sensitivity to mouse P2X₇.