Secretory vesicles from bovine neurohypophysis were reconstituted into lipid bilayers. Electrical measurements on the lipid bilayers under voltage clamp demonstrated the presence of channels that are permeable to chloride ions, are blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and are slightly voltage dependent. When several different membrane fractions were used for the reconstitution, the probability of finding channels correlated with the fraction of secretory vesicle membrane in the membrane fraction, indicating that the secretory vesicles are the source of the channels. The observed anion channel can provide a pathway for the anion transport that has previously been described for secretory vesicles. The secretory vesicle anion channel may play a role in calcium-induced secretion.