Exocytosis of lung surfactant in alveolar type II cells (AT II cells) most likely depends on a subset of membrane-coupled proteins of the SNARE complex. We investigated the significance of syntaxin 3, a t-SNARE protein, for fusion of surfactant storing lamellar bodies (LBs) with plasma membrane by overexpressing syntaxin 3 fused with the green fluorescent protein (Syn3-GFP) in rat AT II cells. Syn3-GFP was found on LB membrane and in the cytoplasm. Using recently described fluorescence techniques (Haller et al., 1998), we examined LB fusion with the plasma membrane after the application of phorbol-12-myristate-13- acetate (PMA, 300 nM) together with calcium ionophore ionomycin (5 ìM). Overexpression of Syn3-GFP significantly inhibited exocytosis after stimulation, as 32% of the Syn3-GFP expressing cells showed exocytosis, compared to 68% of non- infected cells and 76% of cells expressing only GFP. Stimulation with PMA in the absence of ionomycin also resulted in lower response in Syn 3-GFP expressing cells (1%) compared to the response in non-infected cells (13%). Our observations showed that overexpressed Syn3 inhibits exocytosis in AT II cells and thus contribute evidence that Syn3 might be involved in surfactant secretion. Supported by FWF, Projects P15742 & P15743; DFG, Project D1402 and the 6th framework of the European Union, Pulmonet.