As the beta 3-adrenergic receptor (beta3AR) is resistant to short term agonist-promoted desensitization and sequestration, chimeric beta3/beta2 receptors were generated to identify the molecular determinants responsible for these regulatory processes in the beta2AR. By exchanging single or multiple intracellular domains of the beta3AR for the corresponding regions of the beta2AR, we show that specific domains can be identified as additive determinants for desensitization, while sequestration is more dependent on global structural conformation. The carboxyl-terminal tail, the third and the second intracellular loops of the beta2AR provided additive contributions to the desensitization observed upon short term agonist stimulation. The second intracellular loop plays a role which is as important as that of third cytoplasmic loop and carboxyl-terminal tail which had previously been identified as the major determinants of agonist-promoted desensitization. Additive contributions of the cytoplasmic domains of the beta2AR were also observed for agonist-promoted sequestration. The substitution of the first and second intracellular loops and the carboxyl tail were associated with a beta2-like sequestration phenotype. However, in contrast to what is observed for desensitization the co-substitution of the third cytoplasmic loop with any of the other domains completely suppressed sequestration. These results suggest that sequestration depends not only on appropriate interactions of multiple molecular determinants within the cytoplasmic region of the beta2AR but also on conformational determinants that may influence their orientation.