Cytochalasin D, a microfilament disrupting agent, considerably inhibited isoproterenol-stimulated amylase release from enzymatically dispersed parotid acini. Histologically cytochalasin D caused a loss of microvilli lining acinar lumina and luminal enlargement. Nearly empty vacuoles appeared near the luminal and lateral surface, and the membrane bordering on the vacuoles was often continuous with the plasma membrane. Therefore, the vacuolization probably resulted from an elongation of the membrane lining the lumen. Fluorescence staining with rhodamine-phalloidin showed that cytochalasin D caused disruption of microfilaments. When stimulating the cytochalasin D-treated cells with isoproterenol, the number of secretory granules in the cytoplasm diminished markedly and secretory material was observed in the vacuoles, indicating that inhibition of amylase release by cytochalasin D is not due to blocking of exocytosis but to the retention of amylase discharged into vacuoles. These findings suggest that microfilaments are essential in maintaining the parotid acinar structure but do not play a direct part in the movement of secretory granules and their fusion with the luminal membrane.