During the last larval instar of Manduca sexta (tobacco hornworm), the insect enters the wandering phase, in which the animal ceases feeding and the structural reorganization of the midgut begins. Midgut metabolism and ion transport in midguts of feeding and wandering (day 5) fifth-instar tobacco hornworms were compared by measuring midgut short-circuit current (I sc ), enzyme activities, tissue respiration, and mitochondrial oxidation of substrates. The midgut I sc of wandering larvae was 80% lower than the I sc of midguts from younger larvae. The activities of midgut citrate synthase and 3-hydroxyacyl CoA dehydrogenase declined significantly at wandering, indicating that aerobic capacity and fatty oxidation are diminished at this phase of development. This view was supported by the low rate of fatty acyl carnitine oxidation by mitochondria isolated from the midguts of wandering larvae. Although the activity of midgut pyruvate kinase, an index of glycolysis, did not change between feeding and wandering, lactate dehydrogenase activity was highest in the midguts of wandering larvae. This finding indicates that anaerobic glycolysis may play a role in midgut metabolism during wandering. One day prior to wandering there is a significant decline in tissue respiration and mitochondrial succinate oxidation. These metabolic changes may be early indicators of the more dramatic changes in midgut function that are to occur upon reaching the wandering phase. Because changes in midgut function occur abruptly over a 24-hr period prior to wandering, it is argued that midgut function is modulated by the commitment peak of ecdysteroids, which induces the external and behavioral characteristics associated with the wandering phase.