In this work, we studied the in vitro G-protein activation induced by muscarinic agonists using [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) autoradiographic methods to characterize the M(2) and M(4) muscarinic subtypes response. Thus, we describe a detailed characterization of the increases in [(35)S]GTPgammaS binding elicited by carbachol (Cch) and oxotremorine (OXO) (binding in the presence minus binding in the absence of agonist) throughout the gerbil brain (Meriones unguiculatus). For both agonists, the strongest stimulations were found in the superficial gray layer of the superior colliculus, the anteroventral and anteromedial thalamic nuclei, the anterior paraventricular thalamic nucleus, and the caudate-putamen. The comparative study using OXO and Cch suggested that OXO is able to detect differences in the response of structures enriched in M(4) muscarinic receptors, showing a lower potency to stimulate these brain areas. Furthermore, using increasing concentrations of selective M(2) (AF-DX 116) and M(1)/M(4) (pirenzepine) antagonists to inhibit specific Cch- or OXO-induced [(35)S]GTPgammaS binding, significant differences were observed in M(2)-enriched structures but not in M(4)-enriched ones such as the caudate-putamen. These data indicate that appropriate muscarinic agonist stimulation, together with selective inhibition of this effect using functional autoradiography, can be used as a tool to unravel the M(2)- and M(4)-muscarinic subtype-mediated response.