The P2X(1) purinergic receptor subtype occurs on smooth muscle cells of the vas deferens and urinary bladder where it is localized in two different size receptor clusters, with the larger beneath autonomic nerve terminal varicosities. We have sought to determine whether these synaptic-size clusters only form in the presence of varicosities and whether they are labile when exposed to agonists. P2X(1) and a chimera of P2X(1) and green fluorescent protein (GFP) were delivered into cells using microinjection, transient transfection or infection with a replication-deficient adenovirus. The P2X(1)-GFP chimera was used to study the time course of P2X(1) receptor clustering in plasma membranes and the internalization of the receptor following prolonged exposure to ATP. Both P2X(1) and P2X(1)-GFP clustered in the plasma membranes of Xenopus oocytes, forming patches 4-6 microm in diameter. Human embryonic kidney 293 (HEK293) cells, infected with the adenovirus, possessed P2X(1) antibody-labeled regions in the membrane colocalized with GFP fluorescence. The ED(50) for the binding of alpha,beta-methylene adenosine triphosphate (alpha,beta-meATP) to the P2X(1)-GFP chimera was similar to native P2X(1) receptors. ATP-generated whole-cell currents in oocytes or HEK293 cells expressing either P2X(1) or P2X(1)-GFP were similar. Exposure of HEK293 cells to alpha, beta-meATP for 10-20 min in the presence of 5 microM monensin led to the disappearance of P2X(1)-GFP fluorescence from the surface of the cells. These observations using the P2X(1)-GFP chimera demonstrate that P2X(1) receptors spontaneously form synaptic-size clusters in the plasma membrane that are internalized on exposure to agonists.