P2X receptors are cell membrane ion channels gated by extracellular ATP¹. They are widely distributed in vertebrates, having roles in pain and inflammatory processes, but previously unidentified in protozoa. We identified a weakly related gene (p2xA) in the genome of the social amoeba Dictyostelium discoideum, which putatively encoded a P2X receptor with less than 8% identity compared to its vertebrate counterparts. Whole-cell and outside-out patch recordings from HEK293 heterologously expressing p2xA revealed robust desensitising inward currents evoked by ATP (EC₅₀ 176 ± 5 μM) with a unitary conductance of 8.2 pS (-100 mV), demonstrating that the gene encoded a functional P2X receptor. The receptor displayed characteristics of a non-selective cation channel, being permeable to Ca²⁺ (P_Ca/P_Na = 1.5 ± 0.3, n = 8) but relatively impermeable to NMDG (P_NMDG/P_Na = 0.21 ± 0.2, n = 8). The receptor was activated by α,β-methyleneATP, β,γ-methyleneATP, β,γ-imidoATP and benzoyl-benzoylATP (EC₅₀ 147 ± 8, 515 ± 12, 14 ± 3 μM, and > 1 mM respectively), but was insensitive to usual P2X antagonists (PPADS, suramin, TNP-ATP), though blocked by nanomolar copper (IC₅₀ 40 ± 5 nM, n = 5). Despite the weak homology, mutagenesis revealed essential conservation of key structure-function relations; however, some regions deduced to have been critical for ATP binding and/or channel activation in mammalian P2X receptors were clearly not essential². Surprisingly, in D. discoideum, the receptor was found on intracellular membranes, with prominent localisation to an osmoregulatory organelle, the contractile vacuole. Mutant amoeba with a disrupted p2xA gene showed no regulatory cell volume decrease in response to hypotonic stress over a 60 min time period (n = 30 cells). In wild-type cells, regular contractile voiding events occurred with a cycle of 80 ± 5 s (n = 30 cells). In contrast, mutant cells had a reduced frequency of voiding and a prolonged cycle (182 ± 10 s, n = 30 cells). The osmoregulatory defect in mutant cells could be rescued by overexpression of the P2X receptor, and furthermore, the mutant phenotype could be recapitulated in wild-type cells by the addition of copper. These results suggest that loss of the amoeba P2X receptor leads to a compromised contractile vacuole, an inability to osmoregulate, and ultimately cell swelling. These findings demonstrate a hitherto unrecognised role of P2X receptors on intracellular organelles, in this case in osmoregulation.