To study the cell regulation mechanisms of human salivary secretion, surgical specimens of human parotid and submandibular glands were treated in vitro with isoproterenol (beta-agonist), carbachol (muscarinic agonist), and cytochalasin D (microfilament disruptive agent), and morphological changes occurring in serous acinar cells were observed. Control acinar cells treated without secretagogues exhibited only occasional examples of exocytosis. Microfilaments, revealed by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) of F-actin fluorescence stained by rhodamine-phalloidin, were localized underneath the luminal membrane to separate the secretory granules from the luminal membrane. Following isoproterenol treatment, secretory granules made direct contact with the luminal membrane and many omega-shaped exocytotic profiles appeared. TEM and scanning electron microscopy (SEM) showed these profiles to be of granule size or somewhat smaller and to be provided on their cytoplasmic surface with coated pits. Furthermore, CLSM detected the appearance of F-actin fluorescence around the exocytosed granule membranes. Carbachol treatment also evoked the formation in acinar cells of omega-shaped exocytotic profiles some of which were larger than the granules and which exhibited neither coated pits nor associated F-actin fluorescence. To determine if microfilaments regulate the post-exocytotic process of membrane retrieval, we combined isoproterenol treatments with cytochalasin D or carbachol. Following these treatments, F-actin fluorescence surrounding the exocytosed membrane was dispersed or diffused and the exocytotic profiles enlarged remarkably. These results led to the hypothesis that exo/endocytotic processes in human salivary serous acinar cells are regulated differently under autonomic receptor control mediated by microfilaments.