Biochemical data indicate mu-opioid receptor (MOR) coupling predominantly to the G(i) and G(o) family. Additionally, MOR coupling to G(s) is suggested by pharmacological assessments that have revealed excitatory MOR effects, which are resistant to pertussis toxin and sensitive to cholera toxin. However, biochemical evidence for such interactions remains elusive; G(salpha) has not been shown to be present in immunoprecipitate obtained using anti-MOR antibodies. In the current study, the presence of MOR in immunoprecipitate obtained with anti-G(salpha ) antibodies was investigated using Chinese hamster ovary cells stably transfected with MOR (MOR-CHO). MOR Western analyses of opioid naive MOR-CHO membranes immunoprecipitated using anti-G(salpha) antibodies reveal the presence of an approximately 75-80 kDa MOR species. Interestingly, acute and chronic morphine treatment markedly enhances the magnitude of MOR that co-immunoprecipitates with G(salpha), despite the concomitant down-regulation of membrane MOR protein. Enhanced co-precipitation of MOR with G(salpha) occurs without a concomitant increase in the immunoprecipitated G(salpha) protein indicating their increased association. In contrast, chronic morphine diminishes the co-immunoprecipitation of MOR with G(ialpha). Moreover, although only a single MOR species co-immunoprecipitated with G(salpha), MOR Western analysis of MOR-CHO membranes as well as immunoprecipitate obtained with either anti-MOR or anti-G(ialpha) antibodies reveals the presence of multiple molecular mass species of MOR. These data reveal the existence of a subset of MORs whose association with G(salpha) can be enhanced by morphine exposure. Notably, the regulation by chronic morphine of MOR association with G(salpha) and G(ialpha) is reciprocal. The relevance of MOR-Gs(alpha) coupling to opioid tolerance formation is discussed.