Opiates are analgesics of choice in the treatment of chronic pain, but their long-term use leads to the development of physiological tolerance. Thus, understanding the mechanisms modulating the response of their receptor, the µ opioid receptor (µOR), is of great clinical relevance. Here we show that heterodimerization of µOR with opioid receptors (OR) leads to a constitutive recruitment of -arrestin2 to the receptor complex resulting in changes in the spatio-temporal regulation of ERK1/2 signaling. The involvement of -arrestin2 is further supported by studies using -arrestin2 siRNA in cells endogenously expressing the heterodimers. The association of -arrestin2 with the heterodimer can be altered by treatment with a combination of µOR agonist (DAMGO) and OR antagonist (Tipp), and this leads to a shift in the pattern of ERK1/2 phosphorylation to the pattern observed with µOR alone. These data indicate that, in the naive state, µOR-OR heterodimers are in a conformation conducive to -arrestin-mediated signaling. Destabilization of this conformation by cotreatment with µOR and OR ligands leads to a switch to a non--arrestin-mediated signaling. Taken together, these results show for the first time that µOR-OR heterodimers, by differentially recruiting -arrestin, modulate the spatio-temporal dynamics of opioid receptor signaling.--Rozenfeld, R., Devi, L. A. Receptor heterodimerization leads to a switch in signaling: -arrestin2-mediated ERK activation by µ- opioid receptor heterodimers.