A new protein was purified from the pancreatic juice of rats with acute pancreatitis. That protein, not detectable in control animals, was called “pancreatitis-associated protein.” It was first observed 6 hours after induction of experimental pancreatitis with taurocholate or cerulein, reached maximal levels of 45 μg/mg protein in zymogen granules and 1.8 μg/mg protein in pancreatic tissue during the acute phase (48 hours), and disappeared during recovery (day 5). It was never detected in spleen, liver, kidney, heart, or lung. The detection limit of the assay system was 12 ng/mg protein, so that pancreatitis-associated protein levels increased at least 100-fold in pancreatic tissue during the acute phase. The molecular weight (12,000) and isoelectric point (8.2) were determined by two-dimensional gel electrophoresis, Subcellular fractionation and immunoelectron microscopy showed that the protein was synthesized on the rough endoplasmic reticulum and stored in zymogen granules before being secreted, similar to other pancreatic secretory proteins. Immunoblotting and two-dimensional gel electrophoresis revealed that the same protein was synthesized upon induction of pancreatitis by cerulein infusion, by retrograde injection of bile acids, or pancreatitis induced by pancreatic surgery. The pancreatitis-associated protein is therefore an acute-phase protein that differs from other proteins of that family because of its exocrine nature.