The activities of trypsin, aminopeptidase, and alpha-glucosidase were studied in the whole midgut, anterior and posterior midgut, and posterior midgut lumen and epithelium of the mosquito Anopheles stephensi Liston. Trypsin activity was restricted entirely to the posterior midgut lumen. No trypsin activity was found before the blood meal, but activity increased continuously up to 30 h after feeding, and subsequently returned to baseline levels by 60 h. Aminopeptidase was active in anterior and posterior midgut regions before and after feeding. In whole midguts, activity rose from a baseline of approximately 3 enzyme units (EU) per midgut to a maximum of 12 EU at 30 h after the blood meal, subsequently falling to baseline levels by 60 h. A similar cycle of activity was observed in the posterior midgut and posterior midgut lumen, whereas aminopeptidase in the posterior midgut epithelium decreased in activity during digestion. Aminopeptidase in the anterior midgut was maintained at a constant low level, showing no significant variation with time after feeding. alpha-glucosidase was active in anterior and posterior midguts before and at all times after feeding. In whole midgut homogenates, alpha-glucosidase activity increased slowly up to 18 h after the blood meal, then rose rapidly to a maximum at 30 h after the blood meal, whereas the subsequent decline in activity was less predictable. All posterior midgut activity was restricted to the posterior midgut lumen. Depending upon the time after feeding, greater than 25% of the total midgut activity of alpha-glucosidase was located in the anterior midgut. The enzyme distributions are consistent with described structural models for digestion in mosquitoes. After blood meal ingestion, proteases are active only in the posterior midgut. Trypsin is the major primary hydrolytic protease and is secreted into the posterior midgut lumen without activation in the posterior midgut epithelium. Aminopeptidase activity is also luminal in the posterior midgut, but cellular aminopeptidases are required for peptide processing in both anterior and posterior midguts. alpha-glucosidase activity is elevated in the posterior midgut after feeding in response to the blood meal, whereas activity in the anterior midgut is consistent with a nectar-processing role for this midgut region.