Murine models are increasingly used for investigations of sleep, yet no previous studies have characterized cholinergic activation of guanine nucleotide binding proteins (G proteins) in mouse brainstem nuclei known to regulate sleep. This study used in vitro [ 35 S]guanylyl-5′- O -(Γ-thio)-triphosphate ([ 35 S]GTPΓS) autoradiography to test the hypothesis that muscarinic cholinergic receptors activate G proteins in C57BL/6J (B6) mouse brainstem. The nuclei studied are homologous to those known in rat and cat to modulate sleep and nociception. In B6 mouse, carbachol significantly increased specific binding of [ 35 S]GTPΓS in the pontine reticular nucleus, caudal part (79%); pontine reticular nucleus, oral part (131%); laterodorsal tegmental nucleus (56%); pedunculopontine tegmental nucleus (86%); dorsal raphe nucleus (53%); dorsal medial periaqueductal gray (54%); and ventrolateral periaqueductal gray (52%) when compared with basal binding. Carbachol-induced G protein activation was concentration-dependent and blocked by atropine, demonstrating mediation by muscarinic receptors. G protein activation by carbachol was heterogeneous across B6 mouse brainstem nuclei. Comparison of [ 35 S]GTPΓS binding between mouse and rat revealed different magnitudes of G protein activation in the pontine reticular formation. In the same pontine reticular formation area of B6 mouse where in vitro treatment with carbachol activates G proteins, in vivo microinjection of cholinomimetics causes a rapid eye movement sleep-like state. These data provide the first direct measurement of muscarinic receptor-activated G proteins in B6 mouse brainstem nuclei known in other species to regulate sleep. J. Comp. Neurol. 457:175–184, 2003. © 2003 Wiley-Liss, Inc.