Adrenomedullin (ADM) acts as an autocrine or a paracrine factor in the regulation of cardiac function. The intracellular mechanisms involved in the direct effect of ADM on adult rat ventricular myocytes (ARVMs) are still to be elucidated. In ARVMs from normal rats, ADM produced an initial (< 30 min) increase in cell shortening and Ca2+ transients and a marked decrease in both on prolonged incubation (> 1 h). Both effects were sensitive to ADM antagonist ADM-(22-52). Treatment with SQ-22536, an inhibitor of adenylate cyclase, blocked the positive inotropic effect of ADM and potentiated its negative inotropic effect. The negative inotropic effect was sensitive to inhibition by pertussis toxin (PTX), an inhibitor of Gi proteins and KT-5720, an inhibitor of PKA. The observations suggest a switch from Gs-coupled to PTX-sensitive, PKA-dependent Gi coupling by ADM in ARVMs. The ADM-mediated Gi-signaling system involves cAMP-dependent pathways because SQ-22536 further increased the negative inotropic actions of ADM. Also, because ADM is overproduced by ARVMs in our rat model of septic shock, ARVMs from LPS-treated rats were subjected to treatment with ADM-(22-52) and PTX. The decrease in cell shortening and Ca2+ transients in LPS-treated ARVMs could be reversed back with ADM-(22-52) and PTX. This indicates that ADM plays a role in mediating the negative inotropic effect in LPS-treated ARVM through the activation of Gi signaling. This study delineates the intracellular pathways involved in ADM-mediated direct inotropic effects on ARVMs and also suggests a role of ADM in sepsis.