Since P2X₃ receptors mediate fast actions evoked by ATP on nociceptive neurons, much interest is currently focussed on their role in either acute or chronic pain. P2X₃ receptors have certain characteristic properties like rapid onset of receptor desensitization from which they recover very slowly via a process strongly dependent on the extracellular concentrations of calcium and magnesium ions. Using either native receptors of small sensory neurons in culture or expressed by HEK cells, we explored whether recovery from desensitization was dependent on the agonist employed, whether rapid desensitization could be actually modulated by a more subtle type of desensitization evoked by very small agonist concentrations, and whether point mutations of the extracellular domain of the P2X3 receptor could separately determine agonist potency, desensitization and recovery. While the onset of desensitization was similar for various agonists, recovery was largely dependent on the type of agonist used and normally proceded in a non-linear fashion. ATP applied at nM concentrations could produce intense desensitization without prior receptor activation and could modulate fast responses to ATP. Distinct sites of the receptor ectodomain appeared to control agonist activity, desensitization, recovery and dependence on divalent cations. Claryfing the complex properties responsible for P2X₃ receptor desensitization might offer new insights into novel pharmacological approaches to control pain.