gamma-Carboxylated proteins were detected in normal human pancreas by immunohistochemistry with a monoclonal antibody (M3B) specific for gamma-carboxyglutamyl residues. Staining appeared to be localized to the glucagon-secreting alpha-cells in the islets of Langerhans. Consistent with this, sections from a glucagonoma were stained much more intensely with the M3B antibody than those from an insulinoma. A murine alpha-cell line (alphaTC1 Clone 9) was cultured and gamma-carboxylated polypeptides, identified immunologically as prothrombin, protein S and (tentatively) Gas6, were isolated from the intracellular compartment by chromatography on an M3B-coupled resin. As in liver, prothrombin is synthesized by alpha-cells as a gamma-carboxylated zymogen that can be cleaved by ecarin to form an active serine protease that is inhibited by hirudin. The pancreas thus appears to be a novel site of synthesis for certain vitamin K-dependent proteins.