In membranes obtained from mu-opioid receptor (MOR) expressing Chinese hamster ovary (CHO) cells (MOR-CHO), the MOR-selective agonist sufentanil produced a concentration-dependent stimulation of guanosine 5'-O-(3-[35S]thio)triphosphate binding to G(s)alpha that was abolished by blocking MOR with naloxone. This unequivocally demonstrates the long-debated functionality of the previously described association of MOR with G(s)alpha. Several complementary observations indicate the relevance of caveolae to MOR-coupled G(s)alpha signaling. 1) In MOR-CHO membranes, sufentanil stimulated the translocation of G(s)alpha into Triton-insoluble membrane compartments. 2) Sufentanil enhanced the coimmunoprecipitation (co-IP) of G(s)alpha and adenylyl cyclase (AC) with caveolin-1 (a marker for caveolae) from the Triton-insoluble membrane fraction of spinal cord and MOR-CHO. 3) MOR blockade (via naloxone) or G(s) inactivation (via cholera toxin) abolished both the increased trafficking of G(s)alpha into the Triton-insoluble membrane fraction of MOR-CHO and the augmented co-IP from spinal cord membranes of G(s)alpha and AC with caveolin-1. This indicates that these events occurred subsequent to activation of MOR and G(s)alpha. Strikingly, lesser-phosphorylated G(s)alpha, which preferentially couple to MOR (Mol Brain Res 135:217-224, 2005; Mol Pharmacol 72:753-760, 2007; Mol Pharmacol 73:868-879, 2008), are concentrated in caveolae, underscoring their relevance to MOR G(s)alpha signaling. MOR-stimulated trafficking of G(s)alpha and AC into caveolae and the likelihood of increased MOR G(s)alpha coupling within caveolae could suggest that they contain the downstream effectors for MOR G(s)alpha AC signaling.