Osmolality increases with pCO(2) in bioreactors with pH control, and it has been shown that osmolality compensation by decreasing the basal NaCl concentration partially :mitigates the adverse effects of elevated pCO(2) on animal cell growth, protein production, and glycosylation. Thus, measurement of osmolality is important for a complete characterization of the culture environment under elevated pCO(2). However, osmolality measurement may be compromised by CO2 evolution. Freezing point depression and vapor pressure depression osmometry were directly compared for the measurement-bf osmolality in samples at elevated pCO(2) (up to 250 mmHg) and at a variety of pH values (6.7-7.5). More:extensive degassing may be expected with the vapor pressure osmometer due to the smaller sample volume and-larger surface area employed. However, both types of osmometer yielded similar results for all pCO(2), and pH values studied. Moreover, the measured values agreed with osmolality values calculated using a semiempirical model. Further analysis showed that, while sample degassing may result in a large decrease in pCO(2), there is little associated decrease in osmolality. The great majority of total CO2 in solution is present as bicarbonate (HCO3-). Although a small amount of HCO2 is converted to CO2 to compensate for CO2 evolution, further depletion of HCO3- is inhibited by the associated increase in-medium pH and by the need for HCO3- to maintain charge neutrality in solution. This explanation is consistent with the observed similarity in osmolality values for the two types of osmometer. It was also observed that osmolality did not change in samples that were frozen at -20 degrees C for up to 1 year. (C) 2000 John Wiley & Sons, Inc.