In the rod cell of the retina, arrestin is responsible for blocking signaling of the G-protein-coupled receptor rhodopsin. The general visual signal transduction model implies that arrestin must be able to interact with a single light-activated, phosphorylated rhodopsin molecule (Rho*P), as would be generated at physiologically relevant low light levels. However, the elongated bi-lobed structure of arrestin suggests that it might be able to accommodate two rhodopsin molecules. In this study, we directly addressed the question of binding stoichiometry by quantifying arrestin binding to Rho*P in isolated rod outer segment membranes. We manipulated the "photoactivation density," i.e. the percentage of active receptors in the membrane, with the use of a light flash or by partially regenerating membranes containing phosphorylated opsin with 11-cis-retinal. Curiously, we found that the apparent arrestinRho*P binding stoichiometry was linearly dependent on the photoactivation density, with one-to-one binding at low photoactivation density and one-to-two binding at high photoactivation density. We also observed that, irrespective of the photoactivation density, a single arrestin molecule was able to stabilize the active metarhodopsin II conformation of only a single Rho*P. We hypothesize that, although arrestin requires at least a single Rho*P to bind the membrane, a single arrestin can actually interact with a pair of receptors. The ability of arrestin to interact with heterogeneous receptor pairs composed of two different photo-intermediate states would be well suited to the rod cell, which functions at low light intensity but is routinely exposed to several orders of magnitude more light.The hundreds of different types of G-protein-coupled receptors (GPCRs) 2 and their binding partners represent versatile protein families, whose individual members have been modified by nature to accomplish many different functions while preserving a basic structure and activation mechanism (1, 2). GPCRs share a common seven-transmembrane helical structure, which, when activated by ligand or stimuli, binds and activates G-proteins to initiate cell signaling (3). Most GPCRs also share a common mechanism of signal termination, which involves receptor phosphorylation and binding of the protein arrestin. However, the fates of arrestin-bound receptors vary widely depending on the type of receptor and its bound ligand. Arrestin-bound receptors may be internalized and degraded, internalized and recycled, or even initiate Gprotein independent signaling (4).Although detailed structural information on different GPCRs (5-9) and arrestins (10 -13) has been available for some time, basic questions regarding their interaction remain unanswered. In particular, the binding stoichiometry (how many active receptors does a single arrestin bind?) is a matter of debate. Historically, one-to-one binding has always been assumed, because many GPCRs are present at low concentrations on the cell surface (1), or in the case of the photoreceptor rhodopsin, activ...