Magnocellular neurons of the supraoptic nucleus (SON) can differentially control peptide release from the somato/dendritic and axon terminal compartment. Dendritic release can be selectively regulated through activation of intracellular calcium stores by calcium mobilizers such as thapsigargin (TG), resulting in preparation (priming) of somato/dendritic peptide pools for subsequent activity-dependent release. As dynamic modulation of the actin cytoskeleton is implicated in secretion from synaptic terminals and from several types of neuroendocrine cells, we studied its involvement in oxytocin and vasopressin release from SON neurons. Confocal image analysis of the somata revealed that the normally continuous cortical band of F-actin is disrupted after high potassium (K+, 50 mm) or TG (200 nm) stimulation. The functional importance of actin remodelling was studied using cell-permeable actin polymerizing (jasplakinolide, 2 μm) or depolymerizing agents (latrunculin B, 5 μm) to treat SON and neural lobe (NL) explants in vitro and measure high K+-induced oxytocin and vasopressin release. Latrunculin significantly enhanced, and jasplakinolide inhibited, high-K+-evoked somato/dendritic peptide release, while release from axon terminals was not altered, suggesting that high-K+-evoked release in the SON, but not the NL, requires depolymerization of the actin cytoskeleton. TG-induced priming of somato/dendritic release was also blocked by jasplakinolide and latrunculin, suggesting that priming involves changes in actin remodelling.