Mammalian P2X receptors are formed by seven subunits (P2X1 to P2X7) that can arrange as homo- or heterotrimers. P2X7 receptors play an important role in the physiology of the immune system, with key roles in mechanisms such as cytokine release, apoptosis and microbial killing. This receptor is frequently co-expressed in immune cells with another member of the P2X family, the P2X4 receptor. The role of P2X4 receptor in immune cells is not as well understood although its up-regulation in spinal cord microglia, as a result of peripheral nerve injury, contributes to neuropathic pain. We recently provided biochemical and electrophysiological evidence of a structural and functional association between P2X4 and P2X7 receptors (Gou et al. 2007). What is not yet known is whether or not individual subunits interact to form heterotrimeric receptors, or if mature homomeric receptors associate in higher order complexes. Analysis of P2X4 and P2X7 receptors heterologously expressed in HEK293 cells by blue native (BN)-PAGE, show that both form stable trimeric complexes and that P2X4, but not P2X7, also appears to form stable hexamers and nonamers as described previously (Aschrafi et al. 2004). Analysis of P2X4 receptors from bone marrow derived macrophages (BMDMs) showed no band corresponding in size to either trimers or hexamers but instead a predominant band of intermediate size, ~100 kDa larger than predicted for a P2X4 trimer. Following partial dissociation a band corresponding in size to the trimer was seen suggesting that the endogenous P2X4 receptors are forming a complex with other protein(s). Cross-linking of surface receptors in microglia followed by SDS-PAGE and western blot with an anti- P2X4 antibody similarly showed a predominant band that was considerably larger than a trimer. A band of the same size was also recognized by the anti- P2X7 antibody, suggesting that the endogenous complexes contain both P2X4 and P2X7. Further analysis of P2X receptor complexes purified from HEK293 cells are being carried out using atomic force microscopy (AFM).