Although the promiscuous nature of G₁₆ allows it to interact with numerous G protein-coupled receptors, several G_i-linked receptors are incapable of activating phospholipase C via G₁₆. A series of chimeras between Gα₁₆ and Gα_z were constructed and assayed for their ability to mediate receptor-induced stimulation of phospholipase C. Two Gα_16/z chimeras harboring 25 or 44 Gα_z-specific sequences at their C termini (named 16z25 and 16z44) were capable of responding to 14 different G_i-coupled receptors tested, including those that were either unable to associate with Gα₁₆ (melatonin Mel1c) or activate Gα₁₆ weakly (μ-opioid and type 1 somatostatin). Agonist-induced stimulation of phospholipase C was more efficiently mediated (higher maximal and lower EC₅₀ value) by 16z44 than by Gα₁₆. Both 16z25 and 16z44 were also coupled to G_s- and G_q-linked receptors. Incorporation of Gα_z sequence at the N terminus of Gα₁₆ did not further enhance the ability of the chimeras to interact with G_i-coupled receptors. Expression of the various chimeras was verified by immunodetection and functional analysis of their constitutively activated mutants. These results show that the incorporation of α4/β6 and α5 regions of Gα_z into a Gα₁₆ backbone can improve the recognition of G_i-coupled receptors. Gα_16/zchimeras with expanded capability to interact with G_i-linked receptors may be used to link orphan receptors to the stimulation of phospholipase C.