Previously, we demonstrated that the third intracellular (3i) loop of the heptahelical α2A-adrenergic receptor (α2AAR) is critical for retention at the basolateral surface of polarized Madin-Darby canine kidney II (MDCKII) cells following their direct targeting to this surface. Findings that the 3i loops of the D2 dopamine receptors interact with spinophilin (Smith, F. D., Oxford, G. S., and Milgram, S. L. (1999) J. Biol. Chem. 274, 19894-19900) and that spinophilin is enriched beneath the basolateral surface of polarized MDCK cells prompted us to assess whether α2AR subtypes might also interact with spinophilin. [35S]Met-labeled 3i loops of the α2AAR (Val 217-Ala377), α2BAR (Lys 210-Trp354), and α2CAR (Arg 248-Val303) subtypes interacted with glutathione S-transferase-spinophilin fusion proteins. These interactions could be refined to spinophilin amino acid residues 169-255, in a region between spinophilin's F-actin binding and phosphatase 1 regulatory domains. Furthermore, these interactions occur in intact cells in an agonist-regulated fashion, because α2AAR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin may contribute not only to α2AR localization but also to agonist modulation of α2AR signaling.