Muscarine and somatostatin enhance an inward rectifier K + conductance in the AtT-20 pituitary cell line. Both effects are abolished by pertussis toxin (PTX). To determine which PTX-sensitive G protein mediates these agonist effects, we made cDNAs encoding mutant PTX-insensitive G i α subtypes, in which the cysteine residue fourth from the C terminus was replaced with serine. The mutated cDNA was transfected into AtT-20 cells, resulting in stable cell lines overexpressing a G i α subtype. As controls, wild-type G i α cDNA was transfected into AtT-20 cells. The agonist-induced increase of the inward rectifier K + conductance in the transfectants was examined with the whole-cell clamp method. Only in the cell lines into which the mutated (PTX-insensitive) G i 2 α cDNA was transfected, did the muscarine response become PTX-insensitive, suggesting that G i 2 couples to the muscarinic receptor and enhances the activity of the inward rectifier K + channel. However, PTX-insensitive somatostatin responses were not obtained in any of the cell lines transfected with a mutated G i α cDNA, suggesting either that none of the G i subtypes is a transducer for the somatostatin effect or that the mutation prevents the coupling of the G i α to the somatostatin receptor.