1. P2X2 channels are activated by extracellular ATP. Despite being commonly described as non-desensitizing, P2X2 receptors do desensitize or inactivate. In the unspliced, 472 amino acid isoform of the P2X2 receptor, inactivation required membrane disruption and the presence of extracellular Ca2+. 2. The ability to inactivate whole-cell currents developed slowly after breaking in. In contrast, currents from excised patches exhibited rapid (approximately 100 ms) inactivation with a dependence on extracellular Ca2+, ATP and voltage. 3. The inactivation rate increased with the fourth power of [Ca2+] suggesting that the functional channel may be a tetramer. Ca2+ had both a higher affinity and a larger Hill coefficient for inactivation than Mg2+, Ba2+ or Mn2+. Trivalent cations at concentrations up to the solubility product of ATP had no effect. The change in apparent co-operativity with ionic species suggests the presence of experimentally unresolved ligand-insensitive kinetic steps. 4. Based on the weak voltage dependence of inactivation and the lack of effect of intracellular Ca2+ buffers, the Ca2+-binding sites are probably located near the extracellular surface of the membrane. 5. The recovery from inactivation was slow, with a time constant of approximately 7 min. 6. Ca2+-sensitive inactivation only appeared when the membrane was disrupted in some manner. Treatment with actin and microtubule reagents did not induce inactivation, suggesting that an intact cytoskeleton is not necessary. 7. Inactivation rates observed in different patch configurations suggest that the induction of Ca2+-dependent inactivation was due to the loss of a diffusible cofactor located in the membrane or the cytoplasm.