Despite the alarming rate of over 300,000 unintended pregnancies occurring daily, there remain only two available options for male contraceptives. The P2X1 receptor has garnered attention as a genetically validated target for male contraception, offering a promising avenue for research and development.1 However, existing P2X1 receptor antagonists, discovered through high-throughput screening or structure-activity relationship studies, require improvements in both potency and selectivity. My research aims to accelerate drug discovery efforts at the P2X1 receptor by determining the P2X1 receptor structure using cryogenic electron microscopy (cryo-EM) and to use the structure to develop potent P2X1 receptor antagonists. Initial cryo-EM images of the P2X1 receptor unveiled a challenging issue of severe preferred orientation. Through optimisation efforts, we identified a secondary detergent, fluorinated FOS-Choline-8, which proved highly effective in mitigating the orientation bias. These improvements resulted in a 1.96 Å and 2.61 Å structure of the P2X1 receptor in an active ATP-bound and an antagonist NF449-bound state, respectively. We conducted experiments to assess both novel and established P2X1 receptor binders using a HEK293 cell line expressing P2X1 receptors in both calcium mobilization and radioligand binding assays. Single residues of the P2X1 receptor binding site were mutated and tested using calcium mobilisation and radioligand binding assays to reveal the molecular elements responsible for receptor binding to ATP and NF449. Single residue P2X1 receptor mutations produced significant decreases in the EC50 of α,β-methylene ATP and decreases in the IC50 of NF449 compared to WT-P2X1. The next step is to generate novel, and improve current, P2X1 receptor antagonists leveraging our high-resolution P2X1 receptor structures.