P2X purinergic receptors are intensively studied because of their importance in physiological and pathophysiological processes. Seven subunit isoforms are known -P2X1-P2X7. With the exception of P2X6, all of these subunits form homotrimers. In addition, six heterotrimeric channels have so far been identified – P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6 and P2X4/6. Furthermore, an interaction between two homotrimers – P2X4 and P2X7 – has been suggested. Importantly, the existence of this wide variety of assembly states has functional as well as structural implications. Since P2X2 and P2X4 subunits have overlapping expression in various organs, we have investigated the nature of their interaction at the molecular level. tsA 201 cells were co-transfected with P2X2 subunits bearing a green fluorescent protein (GFP) tag and P2X4 subunits bearing a haemagglutinin (HA) tag. We found that P2X2 and P2X4 could be co-immunoprecipitated using either anti-P2X2 or anti-P2X4 antibodies (followed by immunoblotting with either anti-GFP or anti-HA antibodies), indicating that the two subunits interact. Chemical cross-linking of proteins from transfected cells revealed the presence of P2X subunit monomers, dimers and trimers on SDS-polyacrylamide gels. After co-transfection with P2X2 and P2X4, only bands corresponding to homodimers and homotrimers were seen; there was no evidence for the presence of heterodimers or heterotrimers. When cells were co-transfected with His6-tagged P2X2 subunits and HA-tagged P2X4 subunits, both subunits could be isolated through binding of the His6 tag to Ni2+-agarose beads, again indicating an interaction between the two subunits. Proteins were eluted from the Ni2+-agarose beads with imidazole and analysed by atomic force microscopy (AFM). AFM allows us to image single protein molecules, and to compare the measured protein molecular volumes with volumes expected on the basis of a protein’s molecular mass. A characteristic feature of the AFM images of P2X2/P2X4 receptors was the presence of closely apposed pairs of particles, each of which had a molecular volume close to that expected of a P2X trimer. We conclude that P2X2 and P2X4 subunits interact through dimerization of homotrimers, rather than through the generation of P2X2/4 heterotrimeric receptors.