We investigated the role of cortical actin filaments (F-actin) in the regulation of PRL secretion in cultured normal anterior pituitary cells. F-actin dynamics were evaluated by fluorescence microscopy, and PRL secretion from attached cells was measured by the reverse hemolytic plaque assay. F-actin localized to the periphery of lactotropes. PRL-releasing factors such as TRH, vasoactive intestinal peptide (VIP), and forskolin, or removal of the PRL-inhibiting factor dopamine (DA) from cultures chronically exposed to DA, caused fragmentation, i.e. focal disassembly of cortical F-actin. Basal, VIP-, and DA withdrawal-induced cortical F-actin disassembly were dependent on extracellular Ca2+ whereas TRH- and forskolin-induced disassembly were not. Short-term (5 min) treatment of cells with the F-actin-disrupting agent cytochalasin D (CD) enhanced basal PRL secretion but did not further stimulate TRH- or VIP-induced PRL secretion. The results support the existence of a causal link between F-actin disassembly and increased PRL secretion. On the other hand, exposure of cultures to DA decreased the percentage of cells showing cortical F-actin disassembly within minutes. Longer treatments (2-4 h) caused stabilization of cortical actin filaments as revealed by the protection vis-a-vis the depolymerizing effect of CD. The protective effect was specific for lactotropes and was evident with DA concentrations as low as 50 nM. Chronic exposure of the cells to DA blocked CD- and TRH-evoked actin disassembly and PRL secretion while VIP-induced effects were partially inhibited. Stabilization of F-actin with the marine sponge venom, jasplakinolide, also decreased basal and stimulated PRL secretion. In conclusion, our results suggest that, first, the cortical actin cytoskeleton of lactotropes is an integrator of the multiple factors regulating PRL secretion directly on the lactotrope, and second, the tonic inhibition of PRL secretion is mediated, at least in part, by DA-induced stabilization of cortical F-actin.