In neuroblastoma-glioma (NG108-15) hybrid cells, opiates inhibit adenylate cyclase and stimulate a low Km GTPase. It has been postulated that the stimulation of GTPase plays a role in opiate inhibition of adenylate cyclase. Treatment of NG108-15 cells with pertussis toxin attenuates receptor-mediated inhibiton of adenylate cyclase. The toxin acts by catalyzing the ADP-ribosylation of a 41,000-dalton substrate believed to be a part of the receptor-adenylate cyclase complex. We have found that toxin treatment of NG108-15 cells results in inhibiton of the opiate-stimulated GTPase. The concentration of toxin required for inhibition of this GTPase was similar to that needed for both attenuation of opiate inhibition of adenylate cyclase and ADP-ribosylation the 41,000-dalton substrate. Inhibition of the opiate-induced GTPase by pertussis toxin in isolated membranes required NAD, consistent with the hypothesis that this effect of the toxin resulted from ADP-ribosylation of a protein component of the system. Since the opiate-stimulated GTPase is believed to play a role in the receptor-mediated decrease in adenylate cyclase activity, inhibition of this GTPase may be an important part of the mechanism by which toxin interferes with opiate action of an adenylate cyclase.