We have previously suggested that synexin (annexin VII), a Ca²⁺-dependent phospholipid binding protein, may have a role in surfactant secretion, since it promotes membrane fusion between isolated lamellar bodies (the surfactant-containing organelles) and plasma membranes. In this study, we investigated whether exogenous synexin can augment surfactant phosphatidylcholine (PC) secretion in synexin-deficient lung epithelial type II cells. Isolated rat type II cells were cultured for 20-22 h with [³H]choline to label cellular PC. The cells were then treated with -escin, which forms pores in the cell membrane and releases cytoplasmic proteins including synexin. These cells, however, retained lamellar bodies. The permeabilized type II cells were evaluated for PC secretion during a 30-min incubation. Compared with PC secretion under basal conditions, the presence of Ca²⁺ (up to 10 µM) did not increase PC secretion. In the presence of 1 µM Ca²⁺, synexin increased PC secretion in a concentration-dependent manner, which reached a maximum at ~5 µg/ml synexin. The secretagogue effect of synexin was abolished when synexin was inactivated by heat treatment (30 min at 65°C) or by treatment with synexin antibodies. GTP or its nonhydrolyzable analog :-imidoguanosine-5'-triphosphate also increased PC secretion in permeabilized type II cells. The PC secretion was further increased in an additive manner when a maximally effective concentration of synexin was added in the presence of 1 mM GTP, suggesting that GTP acts by a synexin-independent mechanism to increase membrane fusion. Thus our results support a direct role for synexin in surfactant secretion. Our study also suggests that membrane fusion during surfactant secretion may be mediated by two independent mechanisms.