The ATP-gated P2X7 receptors are electrophysiologically unique among the members of the P2X ionotropic receptors family by the fact that they exhibit cationic currents that facilitate when brief agonist applications are repeated. The aim of this study was to elucidate the molecular basis of this facilitation phenomenon recorded by the whole-cell patch clamp configuration in HEK293 cells transiently expressing rat P2X7 receptors. Repeated application of maximum ATP concentration (5 mM, 10 s duration applied at 40 s intervals) resulted in a 2.5-fold increase in current density over the first four applications and then remained at this facilitated level for all subsequent applications (up to 4 min). Facilitation was highly dependent on the intracellular calcium activity and was significantly decreased by the calmodulin-binding peptide (CBP) while co-expression of the dominant-negative calmodulin (CaM1234) was less effective to inhibit facilitation. By searching in a calmodulin target database, we identified an IQ-independent putative calmodulin binding site, made by 17 consecutive amino acid residues from I541 to R557, in the P2X7 cytosolic C-terminus. A double-mutant receptor (I541T, S552C) in this region, generated in order to remove the putative calmodulin binding site, showed no Ca2+/calmodulin dependent facilitation. We also demonstrated by co-immunoprecipitation experiments that calmodulin interacts with the wild-type receptor and that this interaction is increased when receptors are activated by ATP. Calmodulin did not co-immunoprecipitate with the I541T, S552C double-mutant receptor. These results suggest that calmodulin is not pre-bound to the receptor but associates rapidly as the Ca2+ concentration increases in the cell. This study presents, for the first time, evidence that calmodulin associates with a purinergic receptor, the P2X7 receptor, and is responsible for its facilitation.