Opioids are regarded to act via receptors interacting with heterotrimeric pertussis toxin (PTX)-sensitive G proteins. In membranes of SH-SY5Y cells, the mu-selective agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO) and the delta-selective agonist [D-Pen2,Pen5]-enkephalin (DPDPE) stimulated incorporation of the photoreactive GTP analog [alpha-32P]GTP azidoanilide into proteins comigrating with the alpha subunits of G(i1), G(i2), G(i3), G(o1), and another form of G(o), presumably G(o2). In membranes of PTX-treated cells, both agonists were ineffective. Subtype-specific immunoprecipitation of G protein alpha subunits photolabeled in the absence or presence of agonists revealed profound differences between mu and delta opioid receptors in coupling to PTX-sensitive G proteins. Whereas activated delta opioid receptors preferentially coupled to G(i1), activated mu opioid receptors more effectively coupled to G(i3). Additionally, we provide evidence that G(o) subtypes are also differentially activated by the two receptors. Thus, mu and delta opioid receptors appear to discriminate between PTX-sensitive G proteins and lead to activation of distinct G protein subtypes.