Neurosecretory cells, including chromaffin cells, possess a mesh of filamentous actin underneath the plasma membrane. It has been proposed that filamentous actin network separates the secretory vesicles into two compartments: the reserve pool and the release-ready vesicle pool. Disassembly of chromaffin cell cortical filamentous actin in response to stimulation allows the movement of vesicles from the reserve pool into the release-ready vesicle pool. Electron microscopy of cytoskeletons revealed the presence of polygonal areas almost devoid of actin filaments in stimulated cells. The percentage of stimulated cells showing disrupted cytoskeleton correlates well with the increase in secretion in these cells. Fine filaments also remain in these areas of disassembly, and these reacted with actin antibodies, as demonstrated by immunogold staining. In addition, the movement of vesicles between pools requires Ca(2+) and ATP, a condition for activation of a molecular motor. Confocal microscopy images demonstrated colocalization of myosin Va with dopamine-beta-hydroxylase. Cell depolarization induced the dissociation of myosin Va from chromaffin vesicles. 2,3-Butadione-2-monoxime (BDM), an inhibitor of myosin ATPase, inhibited secretion, suggesting a blockage for chromaffin vesicle transport between the reserve pool and the release-ready vesicle pool. On the other hand, myosin II subcellular distribution was not affected by cell depolarization. Confocal microscopy images show myosin II to be localized in the cell cortex and in some perinuclear structures. Chromaffin vesicles were not stained by myosin II antibody.