The effects of changes in osmotic pressure on the production of tissue plasminogen activator (tPA) by Chinese hamster ovary (CHO) cells In suspension were investigated. The osmotic pressure was shifted up from 300 mOsm/kg and down from 400 or 500 mOsm/kg to 400, 350, or 300 mOsm/kg during the cell growth and tPA production phases. In both phases, the specific cell growth rate was higher at a lower osmotic pressure after the shift, whatever it was up or down. After the osmotic pressure was shifted up during the production phase, the specific tPA production rate was high at a high osmotic pressure; however, shifting the osmotic pressure down also resulted in a high specific rate of tPA production. Additionally, specific production rate increased for 2 d after upward or downward shifts during the growth phase. Shifting the osmotic pressure cyclically between 300 and 500 mOsm/kg during the production phase improved the tPA production 1.13-fold compared with the amount produced at a constant osmotic pressure of 300 mOsm/kg.