A gastric serine protease(s) was found in porcine gastric antral mucosa and was shown to be distributed in the endoplasmic reticulum (ER)-microsome fraction and also in the vesicle fraction. Two forms of the protease were purified over 6,000-fold from the ER-microsome fraction. Analyses of various molecular and enzymatic characteristics including the N-terminal and partial internal amino acid sequences of both forms revealed that they share the same properties and are indistinguishable from porcine pancreatic trypsin. This is the first time that trypsin or a protease almost identical with trypsin has been found to be present intracellularly in normal tissues. The gastric trypsin activities from the ER-microsome and the vesicle fractions were located in distinct density regions upon density gradient centrifugation, which indicates association of the protease with different organelle membranes. Taken together, these results suggest that there may be a novel function of trypsin in the gastric mucosa; it might function as a specific degrading or processing enzyme as an intracellular protease.