The alpha 2A-adrenergic receptor (alpha 2AAR) is polarized to the basolateral membrane of Madin-Darby canine kidney cells via direct targeting. Examination of mutant alpha 2AAR reveals that direct delivery is independent of NH2-terminal glycosylation, COOH-terminal acylation, or protein sequences within the large third cytoplasmic loop or COOH-terminal tail. Combined mutation of these structural features also does not perturb alpha 2AAR delivery, suggesting that a three-dimensional structure imparted by non-contiguous endofacial sequences does not confer alpha 2AAR targeting and that motifs in or near the bilayer must be involved in targeting of the alpha 2AAR. Mutation of a conserved Asp residue in transmembrane two that alters receptor-G-protein interactions also does not impair alpha 2AAR targeting. Finally, modification of sequences in transmembrane seven that resemble tyrosine-containing endocytosis motifs utilized for targeting by some proteins does not perturb alpha 2AAR sorting. Interestingly, deletion of the large third cytoplasmic loop of the alpha 2AAR decreases receptor half-life on the basolateral surface from approximately 11 to 4.5 h without altering the ability of the alpha 2AAR to couple to G-proteins. These data suggest that although targeting of the alpha 2AAR likely involves bilayer sequences, the third cytoplasmic loop may contain structural features that promote stabilization of the alpha 2AAR on the basolateral surface of Madin-Darby canine kidney cells.