The spectrum of Tenebrio molitor larval digestive proteinases was studied in the context of the spatial organization of protein digestion in the midgut. The pH of midgut contents increased from 5.2-5.6 to 7.8-8.2 from the anterior to the posterior. This pH gradient was reflected in the pH optima of the total proteolytic activity, 5.2 in the anterior and 9.0 in the posterior midgut. When measured at the pH and reducing conditions characteristic of each midgut section, 64% of the total proteolytic activity was in the anterior and 36% in the posterior midgut. In the anterior midgut, two-thirds of the total activity was due to cysteine proteinases, whereas the rest was from serine proteinases. In contrast, most (76%) of the proteolytic activity in the posterior midgut was from serine proteinases. Cysteine proteinases from the anterior were represented by a group of anionic fractions with similar electrophoretic mobility. Trypsin-like activity was predominant in the posterior midgut and was due to one cationic and three anionic proteinases. Chymotrypsin-like proteinases also were prominent in the posterior midgut and consisted of one cationic and four anionic proteinases, four with an extended binding site. Latent proteinase activity was detected in each midgut section. These data support a complex system of protein digestion, and the correlation of proteinase activity and pH indicates a physiological mechanism of enzyme regulation in the gut.