The involvement of G protein-coupled receptor kinase 2 (GRK2) in the agonist-induced desensitization of delta-opioid receptor-mediated inhibition of cAMP formation in NG108-15 mouse neuroblastomaxrat glioma hybrid cells was investigated. Pretreatment of wild-type cells with the delta-opioid receptor agonist [D-Pen(2,5)]-enkephalin (DPDPE; 100 nM) for as little as 5 min produced marked desensitization of subsequent DPDPE-mediated inhibition of iloprost (300 nM)-stimulated cAMP formation. In NG108-15 cells stably overexpressing wild-type GRK2 or dominant negative mutant GRK2 (DNM GRK2), the DPDPE-induced desensitization of cAMP inhibition was the same as in plasmid-transfected control cells. Pretreatment of wild-type cells with the inhibitors of receptor internalization, concanavalin A (0.25 mg ml(-1)) or hypertonic sucrose (0.4 M), also failed to inhibit DPDPE-mediated desensitization. Finally, in NG108-15 cells stably overexpressing G protein-coupled receptor kinase 6 (GRK6), DPDPE-induced desensitization was significantly increased as compared to plasmid-transfected control cells. These results indicate that GRK2 is unlikely to mediate the desensitization of endogenous delta-opioid receptors in NG108-15 cells, but that other GRKs, such as GRK6, may be more important.