Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with 10 nM [D-Ala2,D-Leu5] enkephalin (DADLE) results in a reduction of cell-surface opiate delta receptors. Whether opiate receptor internalization requires the activation of the guanine nucleotide-binding protein (Ni) is unclear. Hence, activation of Ni was attenuated by treating hybrid cells with 100 ng/ml pertussis toxin (PT) for 3 h, which resulted in a decrease in DADLE's ability to inhibit adenylate cyclase activity. Despite this prior treatment with PT, chronic exposure of these cells to 10 nM DADLE resulted in a time-dependent decrease in both [3H]diprenorphine and [3H]DADLE binding. This reduction in 3H-ligand binding in cells previously treated with PT represented internalization of the receptors because translocation was observed of bound [3H]DADLE from plasma membrane fractions to the lysosomal fractions in the Percoll gradients. Thus, opiate receptors internalize without activation of Ni. The internalization of opiate receptors was not accompanied by Ni. By measuring the amount of the 41-kDa alpha subunit being labeled by PT with [32P]NAD+, it was determined that plasma membrane preparations, of both the control cells and cells treated with 10 nM of DADLE for 4 h, contained equal concentrations of Ni, 2 pmol of Ni/mg of protein. Additionally, there was no measurable alteration in the amount of PT substrate in the lysosomal fractions of the DADLE-treated cells as compared to that of control cells. Chronic DADLE treatment resulted in a decrease in Km value of NAD+ in the ADP-ribosylation of 41-kDa subunit of Ni. In summary, opiate receptors internalize as agonist-receptor complexes without the guanine nucleotide-binding component.