The third intracellular (3i) loops of the α2A- and α2B-adrenergic receptor (AR) subtypes are critical for retention of these receptors at the basolateral surface of polarized Madin-Darby canine kidney (MIICKII) cells at steady state. The third intracellular loops of the α2A, α2B, and α2C-AR subtypes interact with spinophilin, a multidomain protein that, like the three α2-AR subtypes, is enriched at the basolateral surface of MDCKII cells. The present studies provide evidence that α2-AR interaction with spinophilin contributes to cell surface stabilization of the receptor. We exploited the unique targeting profile of the α2B-AR subtype in MDCKII cells: random delivery to apical and basolateral surfaces with rapid (t1/2 ≤ 60 min) apical versus slower (t1/2 = 10-12 h) basolateral turnover. Apical delivery of a spinophilin subdomain containing the α 2-AR-interacting region (Sp151-483) by fusion with apically targeted p75NTR extended the half-life of α2B-AR at the apical surface to ∼3.6 h and eliminated the rapid phase (0-60 min) of α2B-AR turn-over on that surface. Furthermore, we examined α2B-AR turnover at the surface of mouse embryo fibroblasts derived from wild type (Sp+/+) or spinophilin knock-out (Sp -/-) mice. Two independent experimental approaches demonstrated that agonist-evoked internalization of HA-α2B-AR was accelerated in mouse embryo fibroblasts derived from Sp-/- mice. These findings are consistent with the interpretation that endogenous spinophilin contributes to the stabilization of β2B-AR and presumably all three α2-AR subtypes at the surface of target cells and may act as a scaffold that could link α2-ARs to proteins interacting with spinophilin via other domains.