Musca domestica trypsin antibody cross-reacts with polypeptide bands of M(r) 25,000 and 30,000 showing proteolytic activity from Stomoxys calcitrans midgut extracts. Secretory granules from the main enzyme-secreting region, the opaque zone, stained heavily with the trypsin antibody in both unfed and blood-fed flies. Heterogeneous staining of granules suggests the unequal distribution of trypsin in secretory granules. This is also consistent with the occurrence of non-parallel secretion, which is also suggested by the possible preferential release of smaller, heavily stained secretory granules in fed flies. The predigestive, anterior midgut region responsible for rapid dehydration of the blood meal, the reservoir zone, contains a different population of secretory granules which stain heavily with trypsin antibody. This zone contains 20% of the midgut trypsin activity in unfed flies; trypsins are held here as proenzymes which are probably only activated postsecretion. In the midgut lumen of both unfed and blood-fed flies, trypsin is mainly immunolocalized in the ectoperitrophic space. Enzyme assays suggest that 5-15% of the lumenal trypsin is associated with the peritrophic matrix. The finding of intact secretory granules plus cell debris in the ectoperitrophic space of opaque and lipoid zones of blood-fed flies supports the contention that some trypsin is released by apocrine secretion in this insect.