P2X receptors are cation channels gated by extracellular ATP that are formed by the non-covalent assembly of three identical or homologous subunits. All P2X subunits contain ten strictly conserved cysteine residues (denoted here with sequential numbers, C1-C10) in the ectodomain that give rise to five intrachain disulfides per subunit. To examine the role of the cysteine residues in the folding and assembly of the P2X1 receptor, we took advantage of the reducing agent dithiothreitol (DTT) that inhibits formation of disulfides in living cells without perturbing most other functions (Braakman et al. EMBO J. 11, 1717-1722, 1992). in intact X. laevis oocytes, DTT kept newly synthesized P2X1 subunits in their less compact reduced form that aggregated, but did not trimerize or reach the plasma membrane. Upon DTT washout, the preformed P2X1 subunits oxidized into the more compact form and homotrimerized efficiently to form a fully functional ATP-gated receptor that appeared at the plasma membrane. in the mature homotrimeric state, the P2X1 receptor was DTT resistant. The impact of individual cysteine residues on homotrimerization was assessed by studying cysteine-to-serine single mutants. The resulting presence of an odd number of nine cysteine residues in the ectodomain significantly increased aggregate formation. Depending upon which residue was mutated, homotrimerization was impaired (C1S to C6S) or abolished (C7S to C10S), resulting in reduced or absent cell surface expression, respectively. in contrast to the wt rP2X1 receptor, the ectodomain of mutants C1S to C6S contained a maleimide-reactive thiol, indicating invariant accessibility of the unpaired cysteine residue. These results will be discussed in relation to the crystal structure of the zebrafish P2X4 receptor (Kawate et al. Nature 460, 592–599, 2009).