We investigated the role of the actin-based myosin motor, Myosin 5c (Myo5c) in vesicle transport in exocrine secretion. Lacrimal gland acinar cells (LGAC) are the major source for the regulated secretion of proteins from the lacrimal gland into the tear film. Confocal fluorescence and immunogold electron microscopy revealed that Myo5c was associated with secretory vesicles in primary rabbit LGAC. Upon stimulation of secretion with the muscarinic agonist, carbachol, Myo5c was also detected in association with actin-coated fusion intermediates. Adenovirus-mediated expression of GFP fused to the tail domain of Myo5c (Ad-GFP-Myo5c-tail) showed that this protein was localized to secretory vesicles. Furthermore, its expression induced a significant (p0.05) decrease in carbachol-stimulated release of two secretory vesicle content markers, secretory component and syncollin-GFP. Adenovirus-mediated expression of GFP appended to the full length Myo5c (Ad-GFP-Myo5c-full) was used in parallel with adenovirus-mediated expression of GFP-Myo5c-tail in LGACs to compare various parameters of secretory vesicles labeled with either GFP-labeled protein in resting and stimulated LGACs. These studies revealed that the carbachol-stimulated increase in secretory vesicle diameter associated with compound fusion of secretory vesicles that was also exhibited by vesicles labeled with GFP-Myo5c-full was impaired in vesicles labeled with GFP-Myo5c-tail. A significant decrease in GFP labeling of actin-coated fusion intermediates was also seen in carbachol-stimulated LGAC transduced with GFP-Myo5c-tail relative to LGAC transduced with GFP-Myo5c-full. These results suggest that Myosin 5c participates in apical exocytosis of secretory vesicles.