Morphine activates mu-opioid receptors (MORs) without promoting their rapid endocytosis in a number of cell types. A previous study suggested that morphine can drive rapid redistribution of MORs in the nucleus accumbens, but it was not possible in this in vivo study to identify a specific membrane trafficking pathway affected by morphine, to exclude possible indirect actions of morphine via opiate-regulated neural circuitry, or to define the mechanism of this morphine-dependent regulation. In the present study, we addressed these questions using dissociated primary cultures of rat striatal neurons as a model system. Morphine promoted a rapid redistribution of both endogenous and recombinant MORs within 30 min after drug addition to the culture medium. This effect was mediated by rapid endocytosis and occurred in a cell-autonomous manner, as indicated by its detection in cells plated at low density and in cultures in which depolarization was blocked by tetrodotoxin. Morphine-induced endocytosis of MORs was quantitatively similar to that induced by the enkephalin analog D-Ala2-N-Me-Phe4-Glycol5-enkephalin, and endocytosis induced by both ligands was inhibited by a dominant-negative mutant version of arrestin-3 (beta-arrestin-2). These results extend previous in vivo results and indicate that morphine is indeed capable of driving rapid endocytosis of mu-opioid receptors in an important subset of opiate-responsive CNS neurons. They also suggest a cellular mechanism by which beta-arrestins may modulate the physiological effects of morphine in vivo.