The transformation of the parasite Trypanosoma cruzi from the blood-borne trypomastigote to the intracellular amastigote constitutes a key clinical feature in the pathophysiology of Chagas' disease. That this transition occurs without change in the integrity of the plasma membrane of the parasite suggests the presence of biochemical structures, i.e., signal transduction systems, that convey information regarding the external milieu of the host so as to facilitate this transformation. In higher eukaryotes, it has been found that a heterotrimeric GTP-binding protein (G-protein), composed of αβγ subunits, constitutes a critical component of this complex. Two closely related groups of G-proteins are substrates for cholera toxin (CT)- (Gs) and pertussis toxin (PT)- (Gil-3 and Go) dependent ADP ribosylation. In concert, they link plasma membrane receptors to adenylate cyclase, resulting in the stimulation or inhibition, respectively, of cAMP generation. In this report, we demonstrate the presence of both groups of G-proteins. Cholera toxin-dependent ADP ribosylation of 42- and 45-kD proteins was demonstrable in amastigotes (AMAST), in the cytosol of epimastigotes (EPI), and weakly in trypomastigotes (TRYP), suggesting the presence of the stimulatory GTP-binding protein, Gs, in T. cruzi. Antisera generated against the αs subunit of the Gs heterotrimeric protein (anti-αs) bound to a 45-kD protein CT substrate in the rank order TRYP ≫ AMAST ≈ EPI cytosol. Immunoprecipitation of CT-32P-ADP-ribosylated membranes with anti-αs resulted in 42- and 45-kD proteins. However, no Gs-mediated activation of adenylate cyclase was demonstrable in reconstitution studies using cyc- lymphoma cells, which lack a functional Gs but possess a β-adrenergic receptor and adenylyl cyclase enzyme. Pertussis toxin -catalyzed ADP ribosylation was demonstrable in 39–40-kD particulate proteins of EPI, less strongly in AMAST, and least in TRYP, consistent with the presence of inhibitory (Gi) and Go GTP-binding proteins. In support of this observation, immunochemical analysis of the PT substrates identified the presence of αo and αi1-2-3 in EPI, AMAST and TRYP, although, with the exception of αi3, both toxin and associated immunochemical PT substrates are decreased in AMAST and TRYP relative to EPI. Although the functions of these putative G-proteins in T. cruzi are still unclear, their expression may be regulated by the state of parasite differentiation. Despite our inability to demonstrate a function for these G-proteins in the adenylate cyclase signal transduction system, their presence in T. cruzi suggests that they may function in other signal transduction pathways yet to be elucidated. Future investigations of their function may reveal important targets for therapeutic intervention in this disease.