Airway epithelial cells cultured at the air-liquid interface possess highly differentiated functions and structures compared with the cells cultured under immersion. We examined the oxidative metabolism and glycolysis in cow tracheal epithelial cells on Days 3, 6, 10, and 13, cultured under three different conditions: (1) immersion culture on porous filters with apical and basolateral feeding (IM), (2) air-exposed culture on porous filters with basolateral feeding, i.e., air-liquid interface culture (AI), and (3) conventional immersion culture in plastic dishes with apical feeding (DI). Lactate production was less in AI than in IM and DI on Day 3 through Day 13, whereas cellular adenosine triphosphate content and basal O2 consumption were greater. Ouabain-sensitive and ouabain-insensitive O2 consumption, and the uncoupled O2 consumption were also greater in AI. Cytosolic lactate dehydrogenase activities on Day 10 were lower in AI, whereas alpha-ketoglutarate dehydrogenase activities were higher. The increased oxidative metabolism in AI was more pronounced at the late phase of culture (Days 10 and 13). In contrast, glycolysis remained elevated during the experiment in IM and DI. These data suggest that (I) AI begins to promote oxidative metabolism from growth phase by the provision of adequate oxygenation, and then further shifts to oxidative metabolism with differentiation; and (2) apical feeding may be responsible for the disturbance of the development of the oxidative metabolism.