We compared the G-protein requirements for coupling of human and chicken m2 and m4 muscarinic acetylcholine receptors (mAChRs) to inhibition of adenylyl cyclase, using a luciferase reporter gene under the transcriptional control of a cAMP response element as a sensitive monitor of intracellular cAMP levels. Previously, we used this system to demonstrate that the chick m4 receptor preferentially coupled to Gi alpha-2 and G(o)alpha over Gi alpha-1 and Gi alpha-3. We found that both the chick and human m2 mAChRs can couple to Gi alpha-1, Gi alpha-2, Gi alpha-3, and G(o)alpha, while the human m4 mAChR preferentially couples to Gi alpha-2 and G(o)alpha. Both the G(o)1 and G(o)2 forms of the G(o)alpha subunit were effective in reconstituting coupling of the m2 and m4 mAChRs to inhibit adenylyl cyclase activity. The m2 and m4 mAChRs thus couple to inhibition of adenylyl cyclase by overlapping but different sets of G-protein alpha subunits.