Chronic morphine augments protein kinase C (PKC) phosphorylation of G β , which enhances the potency of G βγ to stimulate adenylyl cyclase II (ACII) activity. The present study demonstrates an in vivo association between phosphorylated G β and a specific PKC isoform, PKCγ. We investigated the association of G β and PKCγ by assessing the ability of anti-PKCγ antibodies to co-immunoprecipitate G β from 32 P-radiolabeled Chinese Hamster Ovary cells stably transfected with a μ-opioid receptor (MOR-CHO). PKCγ immunoprecipitate (IP) obtained from MOR-CHO membranes contained radiolabeled signals of ≈33 and 36–38 kDa that were subsequently identified as G β (s). Chronic morphine significantly increased (≈75%) the magnitude of 32 P incorporated into G β present in PKCγ IP. This suggests that G β is an in vivo substrate for PKCγ, which mediates the chronic morphine-induced increment in G β phosphorylation. In order to evaluate AC as a putative effector for phosphorylated G βγ , its presence in IP obtained using anti-AC antibodies was evaluated. Autoradiographic analyses of AC IP also revealed the presence of phosphorylated G β (s), the magnitude of which was significantly enhanced (≈60%) following chronic morphine treatment. This indicates that phosphorylated G βγ associates and presumably interacts in vivo with AC, indicating that it is a target for the enhanced phosphorylated G βγ that is generated following chronic morphine treatment. This would contribute to the previously observed shift from predominantly G iα inhibitory to G βγ stimulatory AC signaling following chronic morphine. The PKCγ–G β –AC complex identified in this study provides an organizational framework for understanding the well-documented participation of PKCγ in opioid tolerance-producing mechanisms.