The objective of this study was to understand the mechanisms involved in P2X₇ receptor activation. Treatments with ATP or with the P2X7 receptor specific ligand BzATP induced pore formation, but the effect was slower in CaSki cells expressing endogenous P2X₇ receptor than in HEK-293 cells expressing exogenous P2X₇ receptor (HEK-293-hP2X7-R). In both types of cells Western blots revealed expression of three forms of the receptor: the functional 85KDa form present mainly in the membrane, and 65KDa and 18KDa forms expressed both in the plasma membrane and in the cytosol. Treatments with ATP decreased transiently the 85KDa form and increased the 18KDa form in the membrane, suggesting internalization, degradation and recycling of the receptor. In CaSki cells ATP stimulated phosphorylation of the 85KDa form on tyrosine and serine residues. Phosphorylation on threonine residues increased with added ATP, and it increased ATP-requirements for phosphorylation on tyrosine and serine residues, suggesting dominant negative effect. In both CaSki and in HEK-293-hP2X7-R cells ATP also increased binding of the 85KDa form to GRK-3, -arrestin-2, and dynamin, and it stimulated -arrestin-2 re-distribution into submembranous regions of the cell. These results suggest a novel mechanism for P2X₇ receptor action, whereby activation involves a GRK-3, -arrestin-2, and dynamin - dependent internalization of the receptor into clathrin-domains, followed in part by receptor degradation as well as receptor recycling into the plasma membrane.