There are ten conserved cysteine residues present in the extracellular loop region of all known mammalian P2X ligand-gated ion channels. In order to investigate the role of these residues in channel function, we mutated individually each cysteine (C) to alanine (A) in the human P2X₁ receptor and assessed their effects in the Xenopus oocyte expression system. The effect of C to A mutation on agonist (ATP) potency was in most cases minimal, with EC₅₀ values ranging from 1 to 5 µM for mutations at cysteines 117, 126, 132, 149, 159, 165, 261 and 270 (wild-type (WT) EC₅₀ for ATP was 1.2 ± 0.2 µM). The effect of mutation at cysteines 217 and 227, however, was more pronounced with EC₅₀ values of 9.7 ± 1.7 and 54.5 ± 16.6 µM, respectively. Peak amplitude of inward currents through mutant channels was similar to WT channels (3-6 µA) except at mutations of cysteines 132, 159, 261 and 270 where current amplitudes were 55.2 ± 9.0, 53.8 ± 4.0, 6.4 ± 2.0 and 4.1 ± 1.0 % of WT responses, respectively. Cell surface biotinylation with sulfo-NHS-LC-biotin revealed the reason for the reduction in current amplitude for the C261A and C270A mutations was an impairment of receptor trafficking to the cell membrane. By effectively moving the cysteine from position 270 to 269 with a double mutation (C270A/H269C), cell surface expression could be partially rescued. However, current amplitudes with this double mutant were smaller than in the single C270A mutation. These results suggest that a disulphide bond forms between cysteines 261 and 270 in the human P2X₁ receptor and that this bond is essential for efficient trafficking to the cell surface. In order to investigate the presence of disulphide bonds in the remaining eight cysteine residues, N-Biotinoylaminoethyl methane-thiosulfonate (MTSEA-Biotin) was used to specifically label free cysteine residues on cell surface receptors. WT receptors did not label with this compound showing that all extracellular cysteines are unavailable, either from being in a disulphide bond or from being in an inaccessible region of the protein structure. Mutation of cysteine at positions 126, 132, 149, 159, 217 and 227 resulted in MTSEA-Biotin labelling, demonstrating that these residues are involved in disulphide bonds as disruption of the bond by removing a cysteine caused the remaining cysteine in the bond to be accessible for labelling. Mutation of cysteines 165 and 117 did not result in MTSEA-Biotin labelling, showing that these two residues are located in an inaccessible area of the protein structure and may or may not be involved in disulphide bonds. In conclusion, there are at least four disulphide bonds out of a possible ten in the extracellular loop of the P2X₁ receptor, and one of these bonds (C261 to C270) is essential for efficient trafficking to the cell membrane.