The involvement of G proteins in the mechanism underlying the increased ANF secretion observed following atrial muscle stretch (stretch-secretion coupling) was assessed using a combined pharmacological, immunocytochemical and tissue fractionation approach. It was found that G_i/o inhibition by pertussis toxin (PTX) abolished stretch-secretion coupling without affecting baseline secretion through a mechanism that is independent of G_q signaling agonists. Mastoparan-7 (MAS-7), a G_i/o agonist, significantly increased ANF secretion even in the absence of muscle stretch through a PTX-sensitive mechanism. By confocal and by electron immunocytochemistry, ANF and G_o partially co-localized while ultracentrifugation analysis suggested the presence of two populations of granules, one of which was partially associated with G_o as demonstrated by Western blotting. PTX did not affect basal or endothelin-1-stimulated ANF secretion, in line with the view that ET-1 signals mainly through G_q. It is concluded there are at least two types of regulated secretory processes in atrial cardiocytes: one is acutely responsive to muscle stretch and is PTX sensitive and the other is Gq-mediated, is PTX insensitive and may be responsible for changes in secretion following chronic changes in the neuroendocrine environment.