ABSTRACT A ligand-independent activator of heterotrimeric brain G-protein was partially purified from detergent-solubilized extracts of the neuroblastoma-glioma cell hybrid NG108-15. The G-protein activator (NG-GPA) increased [35S]GTPgS to purified brain G-protein in a magnesium-dependent manner and promoted GDP dissociation from Goa. The NG-GPA also increased GTPgS binding to purified, recombinant Gia2, Gia3 and Goa, but minimally altered nucleotide binding to purified transducin. The NG-GPA increased GTPgS binding to membrane-bound G-proteins and inhibited basal, forskolin- and hormone-stimulated adenylyl cyclase activity in DDT1-MF-2 cell membranes. In contrast to GPCR-mediated activation of heterotrimeric G-proteins in DDT1-MF-2 cell membrane preparations, the action of the NG-GPA was not altered by treatment of the cells with pertussis toxin. ADP-ribosylation of purified brain G-protein also failed to alter the increase in GTPgS binding elicited by the NG-GPA. Thus, the NG-GPA acts in a manner distinct from that of a G-protein coupled receptor and other recently described receptor-independent activators of G-protein signaling. These data indicate the presence of unexpected regulatory domains on Gi/Go proteins and suggest the existence of pertussis toxin-insensitive modes of signal input to Gi/Go signaling systems. The NG-GPA and related entities provide novel mechanisms for signal input to heterotrimeric G-proteins that may operate independent of a G-protein coupled receptor.