Activation of δ-opioid receptors in NG108-15 cells releases Ca2+ from an intracellular store through activation of a pertussis toxin-sensitive G protein. We tested the hypothesis that activation of δ-opioid receptors mobilizes inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores via liberation of Gβγ. Fura-2-based digital imaging was used to study the mechanism of opioid-induced increases in [Ca2+](i) in NG108-15 cells. Exposure to D-Ala2-D-Leu5 enkephalin (100 nM) for 90 s induced increases in [Ca2+](i) that were blocked by microinjection of the IP3 receptor antagonist heparin (pipette concentration = 100 mg/ml) but not by sham injection. Microinjection of a peptide that binds Gβγ (QEHA, 1 mM) decreased the D-Ala2-D-Leu5 enkephalin-evoked response. Microinjection of an inactive peptide (SKEE, 1 mM) that does not bind to Gβγ failed to inhibit the opioid-induced increase in [Ca2+](i). Microinjection of a peptide (QLKK, 15 mM) that binds to free Gα(q) blocked the increase evoked by 3 nM bradykinin, but microinjection of an inactive peptide (ADRK, 15 mM) did not. Microinjection of QLKK did not significantly affect the opioid- induced increase in [Ca2+](i). Collectively, these data demonstrate that activation of δ-opioid receptors induces the release of Ca2+ from IP(3)- sensitive stores in NG108-15 cells through activation of the βγ subunits of inhibitory G proteins.