Activation of delta-opioid receptors in NG108-15 cells induces the release of calcium from an inositol 1,4,5-trisphosphate- sensitive intracellular store. We used fura-2-based digital imaging to study the effects of prolonged exposure to agonist on opioid-induced increases in [Ca2+]i. Exposure to D-Ala2-E-Leu5 enkephalin (DADLE) (1 microM) for 30 min completely desensitized NG108-15 cells to a second DADLE-induced response. The cells recovered gradually over 25 min following washout of DADLE. The desensitization was not due to depletion of intracellular calcium stores and bradykinin failed to cross-desensitize the DADLE-evoked response, although both agonists mobilized the same Ca2+ store. Desensitization induced by 100 nM DADLE was overcome by a higher concentration of DADLE (100 microM). Treatment with 8-cpt-cAMP (0.1 mM) for 30 min did not influence the DADLE-induced increases in [CA2+]i. Phorbol dibutyrate (PdBu) (1 microM) blocked the response completely. Treatment with the inhibitor of cyclic nucleotide-dependent kinases H8 (1 microM) for 45 min did not prevent DADLE-induced desensitization. Treatment with the protein kinase C (PKC) inhibitors staurosporin (10 nM) and GF-109203X (200 nM) for 45 min reduced desensitization. However, down-regulation of PKC by 24 h exposure to PdBu (1 microM) failed to prevent the DADLE-induced desensitization in NG108-15 cells. Thus, we conclude that multiple pathways participated in desensitization of delta-receptor-mediated Ca2+ mobilization, one of which includes PKC.