Insulin and β-adrenergic agonists stimulate a rapid phosphorylation and sequestration of the β2-adrenergic receptors (β2ARs). Although the expectation was that a common pathway would be involved in the trafficking of the β2AR in response to either hormone, studies reported herein show the existence of unique cytoskeletal requirements for internalization/recycling of G-protein-coupled receptors, such as the β2AR. Treatment of human epidermoid carcinoma A431 cells with nocodazole, which binds tubulin monomer in vivo and catalyzes the depolymerization of microtubules, effectively blocks β-adrenergic agonist-induced, but not insulin-induced, sequestration of β2ARs. Treatment with latrunculin-A, an agent that sequesters actin monomer and leads to loss of actin filaments, had no effect on the ability of β-adrenergic agonists to stimulate internalization of β2ARs, but blocked the ability of insulin to stimulate counterregulation of β2ARs via internalization. Although nocodazole had no effect on insulin-stimulated sequestration of β2ARs, the recycling of the internalized receptors to the cell membrane was sensitive to depolymerization of microtubules by this agent. Latrunculin-A, by contrast, blocks the recycling of β2ARs internalized in response to β-agonist, while attenuating recycling of receptors internalized in response to insulin stimulation. These data show the existence of unique cytoskeletal requirements for G-protein-coupled-receptor trafficking in response to agonist compared with a counterregulatory hormone, and for sequestration versus recycling of the receptors to the cell membrane.