Muscarinic m4 acetylcholine receptors are normally coupled through Gi to inhibition of adenylyl cyclases. In the olfactory bulb and some cultured cells, however, m4 receptors can couple to stimulation of adenylyl cyclase activity. In this study, m4 receptors and specific isozymes of adenylyl cyclases were coexpressed in HEK-293 cells to characterize the mechanism(s) for m4 receptor stimulation of adenylyl cyclases. The calmodulin-sensitive type I and type III adenylyl cyclases were chosen for this study because neither enzyme is stimulated by the beta/gamma complex of G coupling proteins. M4 receptors exhibited either inhibition or stimulation of type I and III adenylyl cyclases depending upon receptor density and agonist concentration. Inhibition of adenylyl cyclase was apparently due to M4 coupling through Gi. Adenylyl cyclase stimulation through m4 receptors was not due to increases in intracellular Ca2+ and stimulation of the calmodulin-sensitive enzymes since it was evident in isolated membranes in the absence of free Ca2+ and with whole cells preloaded with the Ca2+ chelator BAPTA/AM. Stimulation of adenylyl cyclase activities by m4 receptors was apparently mediated via Gs since it was GTP-dependent, was insensitive to pertussis toxin, and was not due to beta/gamma stimulation. Synthetic peptides derived from a G protein activating region of the m4 receptor mimicked the m4-mediated stimulation of adenylyl cyclase activity. These data demonstrate a novel mechanism for muscarinic regulation of adenylyl cyclases that may involve crossover from inhibitory to stimulatory G protein coupling.