The lepidopteran midgut is a model for the oxygen-dependent, electrogenic K+ transport found in both alimentary and sensory tissues of many economically important insects. Structural and biochemical evidence places the K+ pump on the portasome-studded apical plasma membrane which borders the extracellular goblet cavity. However, electrochemical evidence implies that the goblet cell K+ concentration is less than 50 mM. We used electron probe X-ray microanalysis of frozen-hydrated cryosections to measure the concentration of Na, Mg, P, S, Cl, K, Ca and H2O in several subcellular sites in the larval midgut of Manduca sexta under several experimental regimes. Na is undetectable at any site. K is at least 100 mM in the cytoplasm of all cells. Typical in vivo values (mM) for K were: blood, 25; goblet and columnar cytoplasm, 120; goblet cavity, 190; and gut lumen, 180. The high K concentration in the apically located goblet cavity declined by 100 mM under anoxia. Both cavity and gut fluid are Cl deficient, but fixed negative charges may be present in the cavity. We conclude that the K+ pump is sited on the goblet cell apical membrane and that K+ follows a nonmixing pathway via only part of the goblet cell cytoplasm. The cavity appears to be electrically isolated in alimentary tissues, as it is in sensory sensilla, thereby allowing a PD exceeding 180 mV (lumen positive) to develop across the apical plasma membrane. This PD appears to couple K+ pump energy to nutrient absorption and pH regulation.