An immunological approach was used to elucidate whether trypsin is synthesized and stored as trypsinogen in the midgut gland of the shrimp Litopenaeus vannamei. Two peptides were constructed using sequences deduced from known shrimp genes: trypsinogen activation peptide and an internal sequence. These peptides were used as haptens to elicit antibodies in rabbits. Specific antibodies were used to detect trypsinogen by Western blot and in histological sections of the midgut gland. Trypsinogen was found by Western blot and was localized into the midgut gland B cells by using immunohistology. In fed shrimp, trypsinogen associated with food particles was found in the lumen of the midgut gland tubules as well. Our results show that regulation of shrimp trypsin activity is similar to that of frequent feeder species, in which trypsin is stored as a zymogen, waiting for secretion and activation.