The effect of high osmotic pressure on the flux changes of two energy metabolisms from glucose and glutamine by AFP-27 hybridoma cells producing monoclonal antibody (MAb) was investigated in batch cultures at various osmotic pressures in the range from 300 to 424 mOsmol/kg. The specific production rate of MAb (q(MAb)) increased monotonically with increasing osmotic pressure. The specific consumption rates of glucose (nu(G)) and glutamine (nu(GLN)) also increased with the osmotic pressure up to 410 mOsmol/kg. However, there were marked changes in the metabolic fluxes when the osmotic pressure was raised further, resulting in a decrease in nu(G) while nu(GLN) continued to increase. These changes in the metabolic fluxes at osmotic pressures higher than 410 mOsmol/kg were associated with an increased yield of lactic acid from glucose (Y-L/G), which indicated the energy yield from glucose declined at osmolarities higher than 410 mOsmol/kg. On the other hand, because of a larger increment in the specific production rate of ammonia under high osmotic pressure, the yield of ammonia from glutamine (Y-A/GLN) increased monotonically with the osmotic pressure throughout the range 300 to 424 mOsmol/kg, signifying a rise in the energy yield from glutamine. Consequently, the higher specific ATP production rates from glucose and glutamine associated with the flux changes under high osmotic pressure could be one of the reasons for the increase in q(MAb) observed at high osmotic pressures.