The cortical F-actin cytoskeleton represents a negative control for secretion, and it must be locally disassembled to allow chromaffin vesicle exocytosis. Recombinant scinderin (a Ca 2+-dependent F-actin-severing protein) potentiated Ca 2+-evoked F-actin disassembly and exocytosis in permeabilized chromaffin cells, an effect blocked by peptides Sc-ABP 1 and Sc-ABP 2 (with sequences corresponding to two actin-binding sites of scinderin), exogenous γ-actin, or phosphatidylinositol 4,5-bisphosphate (PIP 2). PIP 2 effect was blocked by peptide Sc-PIP 2BP (with sequence corresponding to a PIP 2-binding site of scinderin). Truncated scinderin 254–715 (lacking actin-severing domains) did not potentiate exocytosis. Sc-ABP 1, Sc-ABP 2, and γ-actin also inhibited exocytosis in the absence of recombinant scinderin, suggesting an inhibition of endogenous scinderin. Results suggest that scinderin-evoked cortical F-actin disassembly is required for secretion and that scinderin is an important component of the exocytotic machinery.