Abstract: The effects of lactate and osmolarity on the growth, metabolism and EPO expression of recombinant CHO cells were investigated by pulse cultures with different lactate concentrations and osmolarity.It was demonstrated that lactate molecule itself had little effect on the cell culture.With the increase of lactate concentration from 0 to 69mmol·L ^-1 ,corresponding to the increase of osmolarity from 300 to 420mOsm,the specific growth rate of recombinant CHO cells was reduced by 33%,the specific consumption rates of glucose and glutamine both increased by 23%,the yields of cell growth to glucose consumption and glutamine consumption were reduced by 45% and 47%,respectively,and the specific production rates of lactate almost kept constant,showing that more nutrient was consumed via aerobic metabolism pathway for more energy production to meet the increasing demand of maintaining energy of recombinant CHO cells at high osmolarity.The yield of ammonia production to glutamine consumption increased with the increase of osmolarity,while the yield of alanine was reduced.It was showed that the flux of glutamine consumed via glutamate dehydrogenase pathway increased in response to the increase of osmolarity.On the other hand,EPO specific production rates decreased with the increase of osmolarity because of lower specific growth rate.