P2X receptors are a distinct family of ATP-gated trimeric cation channels involved in a wide array of physiological processes. Recent advances have been made to understanding the molecular structure and function of these ion channels, including the generation of crystal structures of zebrafish P2X4 in both ATP-free and ATP-bound states. However, the structures give snapshots of the conformation of the receptor and do not address the time-course of conformational changes following agonist binding, channel opening, desensitization or agonist unbinding. To address these questions we used voltage clamp fluorometry to study human P2X1 receptors by introducing single cysteine point mutations along the subunit interface at residues around the cysteine rich head region (K138C, E181C), adjacent to the agonist binding site (N284C, K190C) and linking the binding site to the transmembrane portions (D320C, P196C, I62C). Voltage clamp fluorometry allows the simultaneous measurement of channel current and the fluorescence of labelled mutant cysteine residues to detect changes in receptor conformation. In this study cysteine mutant human P2X1 receptors were expressed in Xenopus oocytes and labelled with 2-((5(6)-Tetramethyl-rhodamine)carboxylamino)ethyl Methanethiosulfonate (MTS TAMRA). MTS TAMRA labelled each of the single cysteine point mutants detailed above but did not label the wild type P2X1 receptors. Additionally labelling with MTS TAMRA had no effect on agonist evoked currents, showing that binding of the fluorescent probe did not inhibit channel function. ATP application had little or no effect on fluorescence at D320C, P196C, I62C and E181C mutants however decreases in fluorescence were recorded at K138C, N284C and K190C mutants (approximately 5%, 10% and 15% decrease respectively). These decreases in fluorescence returned to baseline levels following removal of ATP, however the time-course of this recovery appeared dependent on the cysteine mutant. This suggests that there are temporally distinct conformational changes in different parts of the receptor.