In cultured noradrenergic neurons from the rat locus coeruleus, application of recombinant G protein beta 1 gamma 2 subunits (30 nM) to the cytoplasmic side induced single channel activity similar to the somatostatin-induced single channel activity of G protein-coupled inward rectifier potassium channels (Kir (G)). In contrast, recombinant GTP gamma S-activated, myristoylated alpha i2 (100 nM) did not activate this brain Kir (G). Application of beta 1 gamma 2 C68S (30 nM or 150 nM), in which the cysteine residue fourth from the carboxyl terminus of gamma 2 was replaced by serine, failed to activate the brain Kir(G). This mutant lacks prenylation which is required for the association of beta gamma subunit with the cell membrane. Thus, our results suggest that the association of beta gamma subunit with the cell membrane is a prerequisite for activating Kir(G) channels.