The IGF-II/mannose 6-phosphate (M6P) receptor is a single-transmembrane domain glycoprotein that plays an important role in the intracellular trafficking of lysosomal enzymes and endocytosis-mediated degradation of IGF-II. The receptor may also mediate certain biological effects in response to IGF-II binding by interacting with G proteins. However, the nature of the IGF-II/M6P receptor's interaction with the G protein or with G protein-coupled receptor (GPCR) interacting proteins such as β-arrestin remains unclear. Here we report that [¹²⁵I]IGF-II receptor binding in the rat hippocampal formation is sensitive to guanosine-5′-[γ-thio]triphosphate, mastoparan, and Mas-7, which are known to interfere with the coupling of the classical GPCR with G protein. Monovalent and divalent cations also influenced [¹²⁵I]IGF-II receptor binding. The IGF-II/M6P receptor, as observed for several GPCRs, was found to be associated with β-arrestin 2, which exhibits sustained ubiquitination after stimulation with Leu²⁷IGF-II, an IGF-II analog that binds rather selectively to the IGF-II/M6P receptor. Activation of the receptor by Leu²⁷IGF-II induced stimulation of extracellular signal-related kinase 1/2 via a pertussis toxin-dependent pathway. Additionally, we have shown that IGF-II/M6P receptors under normal conditions are associated mostly with detergent- resistant membrane domains, but after stimulation with Leu27IGF-II, are translocated to the detergent-soluble fraction along with a portion of β-arrestin 2. Collectively these results suggest that the IGF-II/M6P receptor may interact either directly or indirectly with G protein as well as β-arrestin 2, and activation of the receptor by an agonist can lead to alteration in its subcellular distribution along with stimulation of an intracellular signaling cascade.