The regulated secretory pathway is activated by elevated cytoplasmic Ca, 2+-however, the components mediating Ca2+ regulation have not been identified. In semi-intact neuroendocrine cells, Cap+-activated secretion is ATP-and cytosol protein-dependent. We have identified a novel brain protein,~ 145, as a cytosolic factor that reconstitutes Ca2+-activated secretion in two neuroendocrine cell types. The protein is a dimer of 145 kd subunits, exhibits Ca’+-dependent interaction with a hydrophobic matrix, and binds phospholipid vesicles, suggesting a membrane-associated function. A pl45-specific antibody inhibits the reconstitution of Cap+-activated secretion by cytosol, indicating an essential role for~ 145. The restricted expression of~ 145 in tissues exhibiting a regulated secretory pathway suggests a key role for this protein in the transduction of Ca2+ signals into vectorial membrane fusion events.