P2X receptors are ATP-gated ion channels with a number of roles in humans and other vertebrates. To understand the full range of physiological functions served by this receptor family, we are investigating P2X receptors in model invertebrates. Hydra, a member of the cnidarian phylum, is thought to be a modern proxy for the first organisms to have evolved a defined nervous system. Considering the important physiological and pathophysiological functions of P2X receptors in higher organisms, understanding P2X receptor signaling in non-mammalian phyla may allow us to uncover new functions that the family serves and further understand the evolutionary origins of purinergic signaling. A P2X receptor from Hydra vulgaris was bioinformatically identified and subsequently cloned and sequenced. Following heterologous expression in human embryonic kidney 293 cells of a synthesized construct of the P2X receptor, with codon usage matched to human usage, we have used whole-cell voltage-clamp electrophysiology to study its pharmacology. Extracellular ATP application results in a concentration-dependent increase in inward current with a mean EC50 of 123.8 ± 21.8 μM (n = 8 cells; SEM) with a Hill coefficient of 1.3. AMP, ADP, dATP, UTP, α,β-methylene ATP and β,γ-methylene ATP all failed to evoke currents at 1 mM. The broad-spectrum antagonists PPADS and suramin (both at 0.1 mM) had little or no effect on ATP-induced currents, with the compounds Brilliant Blue G and Phenol Red also having no effect. Co-application of the P2X4 receptor modulator ivermectin at 3 μM with 0.1 mM ATP potentiated currents by approx. 40%. Ion substitution experiments reveal that the receptor is permeable to K+ ions (PK/PNa = 1.75 ± 0.22, n = 11) and Ca2+ (PCa/PNa = 1.64 ± 0.18, n = 6), but largely impermeable to the inorganic cations Tris+ (0.24 ± 0.03, n = 6) and NMDG+ (0.08 ± 0.02, n = 8). The receptor is Cl- impermeable (PCl/PNa = 0.02 ± 0.007, n = 9). From this, the Hydra P2X receptor displays a rank order of permeability to ions as follows: K+≥Ca2+>Na+>Tris+>>NMDG+>Cl-, consistent mammalian homologues. A custom polyclonal antibody was raised against a peptide corresponding to part of the Hydra P2X receptor C-terminus. Whole polyp immunohistochemistry using the antibody suggests that the receptor is expressed in the phylum-specific nematocytes. Specifically, the P2X receptor appears to be localised to the nematocyst capsule wall and not to the harpoon-like inner structure used to capture prey, or to the nematocyte cell in which the nematocyst is embedded. The internal pressure within the nematocyst capsule used to expel the internal harpoon is in the MPa range (Nüchter et al., 2006) and we hypothesize that the P2X receptor may play a role in the osmotic potential that underlies this. The current focus of the study is to investigate this hypothesis through RNAi mediated knockdown of the receptor.