The GPI-anchored membrane dipeptidase is the major peptidase activity of the secretory granule membrane in the exocrine pancreas. The enzyme is also found in the granule content and in pancreatic secretions. Immunocytochemical localization confirmed its location in the granule membrane and in the acinar cell apical plasma membrane. In the endoplasmic reticulum and Golgi, membrane dipeptidase was strictly membrane-bound. There was no membrane dipeptidase in duct cells. The release of membrane dipeptidase from the membrane starts in the immature granule. To identify the mechanism responsible for its release, secretions were collected from cannulated conscious pig under basal conditions and atropine perfusion. The latter treatment caused complete inhibition of protein secretion but had a negligible effect on membrane dipeptidase activity in the secretions. In secretions, membrane dipeptidase partitioned into the detergent-rich phase on phase separation in Triton X-114, whereas treatment with bacterial phosphatidylinositol-specific phospholipase C caused the peptidase to partition into the aqueous phase, indicating that the secreted enzyme could come from shedding of membrane fragments at the apical surface or via the action of a previously characterized phospholipase A activity.