AbstractThe subcellular distribution of four enzymes (glucose‐6‐phosphatase, phosphodiesterase I, NADPH‐cytochromecreductase, andp‐nitroanisoleO‐demethylase) in the midgut of “wandering” fifth‐instar larvae of the tobacco hornworm,Manduca sexta(L), was determined and the composition of mitochondrial and microsomal pellets was examined by electron microscopy. Most of the glucose‐6‐phosphatase activity and one‐third of the phosphodiesterase I activity were found in the high‐speed supernatant. NADPH‐cytochromecreductase activity was marginal andO‐demethylase activity was undetectable in the supernatant. The highest specific activities for phosphodiesterase I, NADPH‐cytochromecreductase, andp‐nitroanisoleO‐demethylase were measured in microsomes, but the relative specific activity of phosphodiesterase I was only half that obtained with the latter two enzymes. In all subcellular preparations the relative specific activities of NADPH‐cytochromecreductase andp‐nitroanisoleO‐demethylase were closely correlated. It is concluded that glucose‐6‐phosphatase and phosphodiesterase I are not microsomal marker enzymes in the midgut, but the activities of NADPH‐cytochromecreductase andp‐nitroanisoleO‐demethylas