The role of conserved polar glutamine, asparagine and threonine residues in the large extracellular loop, and glycosylation, to agonist action at human P2X1 receptors was tested by generating alanine substitution mutants. For the majority of mutants (Q56A, Q95A, T104A, T109A, Q112A, Q114A, T146A, N153A, T158A, N184A, N191A, N242A, N300A) alanine substitution had no effect on ATP potency. The mutants Q95A, Q112A, Q114A and T158A showed changes in efficacy for the partial agonists BzATP and Ap5A, suggesting that these polar residues may contribute to the gating of the channel. The mutants T186A, N204A and N290A had six-, three- and 60-fold decreases in ATP potency, respectively. For T186A and N290A, the partial agonists BzATP and Ap5A were no longer agonists but still bind to the receptor as shown by the ability to modulate the response to co-applied ATP. N153, N184 and N242 are glycosylated in the endoplasmic reticulum and N300 acquires complex glycosylation in the golgi. These results aid in refining a model for ATP binding at the P2X1 receptor where the residues F185T186, and the conserved triplet N290F291R292, are likely to play a role in ATP action at the receptor.