P2X1 and P2X4 receptors have important roles in the cardiovascular system, including contributions to blood clotting and blood pressure control. Therefore subtype selective P2X antagonists have therapeutic potential for treatment of cardiovascular disease. The mechanism of antagonist binding at P2X receptors is currently unclear. NF449 is a potent antagonist at the human P2X1 receptor (hP2X1) and has an IC50 of ~1nM. The rat P2X4 (rP2X4) receptor is insensitive to NF449 up to concentrations of 100µM. The difference in antagonist sensitivity between hP2X1 and rP2X4 subtypes can be used to investigate the molecular basis of antagonism. In this study chimeras have been generated between the two cardiovascular P2X receptors to replace regions of one receptor with corresponding residues from the other. The effects of these mutations on NF449 potency have then been observed. Initially four chimeras were generated, A,B,C and D which split the ligand binding extracellular loop into four sections of approximately equal size. Each of these sections incorporated a section of the residues surrounding the ATP binding pocket, a site at which antagonist binding has been implicated. The chimeras were expressed in Xenopus oocytes and two electrode voltage clamp recordings performed. Replacing residues 56-133 (chimera A) and 261-324 (chimera D) of the NF449 sensitive hP2X1 receptor with residues of insensitive rP2X4 had no significant effect on NF449 potency. Therefore the variant residues within these sections of the extracellular loop do not account for the difference in NF449 action. When residues 136-181 of hP2X1 were replaced with those of rP2X4 (chimera B), antagonist sensitivity was reduced ~130 fold compared to WT hP2X1. Region 136-181 therefore plays a role in the antagonist sensitivity of the hP2X1 receptor. Within this region four positive charges at positions 136-140 have previously been shown to be involved in NF449 action. Reintroducing these charges to chimera B gave nanomolar NF449 potency, highlighting their importance. Neither making the reciprocal chimera (hP2X1 into rP2X4), nor introducing the four charges alone, gave antagonist sensitivity to the insensitive rP2X4 receptor. Other residues within the receptor must therefore be needed for NF449 to inhibit the current. Replacing residues 184-261 of hP2X1 with those of the rP2X4 receptor (chimera C) also caused a significant ~130 fold decrease in NF449 potency. Residues within this region are therefore likely to be involved in the inhibition of the hP2X1 receptor by NF449. This fits with a model of NF449 binding to the four positive residues and an area within chimera C. In on-going work, sub-chimeras are being made within this region to find specific residues/regions that the NF449 molecule may be interacting with.