The mu-opioid receptor has recently been shown to stimulate phosphoinositide-specific phospholipase C via the pertussis toxin-sensitive G16 protein. Given the promiscuous nature of G16 and the high degree of resemblance of signaling properties of the three opioid receptors, both delta- and kappa-opioid receptors are likely to activate G16. Interactions of delta- and kappa-opioid receptors with G16 were examined by coexpressing the opioid receptors and G alpha16 in COS-7 cells. The delta-selective agonist [D-Pen2,D-Pen5] enkephalin potently stimulated the formation of inositol phosphates in cells coexpressing the delta-opioid receptor and G alpha16. The delta-opioid receptor-mediated stimulation of phospholipase C was absolutely dependent on the coexpression of G alpha16 and exhibited appropriate ligand selectivity and dose dependency. Similar transfection studies revealed only weak stimulation by the mu-opioid receptor, whereas the kappa-opioid receptor produced moderate phospholipase C activity. G alpha16 thus appeared to interact differentially with the three opioid receptors. Radioligand binding assays indicate that the mu-opioid receptor was expressed at a lower level than those of the delta- and kappa-opioid receptors. To examine if differential coupling to G alpha16 is prevalent, a panel of Gs- or Gi-coupled receptors was coexpressed with G alpha16 in COS-7 cells and assayed for agonist-induced stimulation of phospholipase C. Activation of alpha2- and beta2-adrenergic, dopamine D1 and D2, adenosine A1, somatostatin-1 and -2, C5a, formyl peptide, and luteinizing hormone receptors all resulted in stimulation of phospholipase C, with maximal stimulations ranging from 1.5- to almost 17-fold. These findings suggest that the promiscuous G alpha16 can in fact discriminate among different receptors and that such preferential interaction might in part be due to the abundance of receptors.