We have investigated the transduction pathways mediating the contractile effect of two glucagon-containing peptides, glicentin (GLIC) and oxyntomodulin (OXM), on smooth muscle cells isolated from rabbit antrum. Low concentrations of GLIC induced a biphasic and rapid (first phase at 5-8 sec) Ins(1,4,5)P3 production. By comparison, higher concentrations of OXM or OXM(19-37) were required to obtain biphasic time-courses of Ins(1,4,5)P3 production. In a Ca2+ free medium, the first phase of Ins(1,4,5)P3 production induced by GLIC or OXM was maintained, while the second phase disappeared. In saponin-permeabilized cells, all three peptides induced cell contraction with similar efficacies and potencies. Exogenous Ins(1,4,5)P3 mimicked the contractile effect of the peptides and heparin, which inhibits the Ins(1,4,5)P3 binding to its receptor, prevented contraction stimulated by each effector. We conclude that a Ca2+ mobilization from the intracellular stores is essential in the contractile effects of GLIC and OXM. Using the fluo-3 probe, a [Ca2+]i increase was observed in the presence of GLIC, OXM, or OXM(19-37). The three peptides reduced by 30-40% the cAMP content of cells stimulated by forskolin. This effect was pertussis toxin sensitive as demonstrated with OXM(19-37). Our data constitute important clues for the existence in smooth muscle cells of receptor(s) specific for the GLIC/OXM hormones, coupled via G protein(s) to both Ca2+ and cAMP pathways.