Carbon dioxide is a by-product of mammalian cell metabolism that will build up in culture if it is not removed from the medium. Increased carbon dioxide levels are generally not a problem in bench-top bioreactors, but inhibitory levels can easily be reached in large-scale vessels, especially if the aeration gas is enriched in oxygen. Due to the equilibrium attained between dissolved CO2 and bicarbonate, increased pCO2 is associated with increased osmolality in bioreactors with pH control. While a few preliminary reports indicate that elevated pCO2 levels can inhibit cell growth and/or recombinant protein production, no comprehensive analysis of the interrelated effects of elevated pCO2 and osmolality has been published. We have examined the effects of 140, 195, and 250 mm Hg (187, 260, and 333 mbar, respectively) pCO2 (with and without osmolality control) on the growth of and recombinant tPA production by MT2-1-8 Chinese hamster ovary (CHO) cells. The effects of elevated osmolality were also investigated at the control pCO2 of 36 mm Hg. Elevated pCO2 at 310 mOsm/kg osmolality inhibited cell growth in a dose-dependent fashion, with a maximum decrease of 30% in the specific growth rate (μ) at 250 mm Hg. Osmolality alone had no effect on μ, but the combination of elevated pCO2 and osmolality increased the maximal reduction in μ to 45%. Elevated pCO2 at 310 mOsm/kg osmolality decreased the specific tPA production rate (qtPA) by up to 40% at 250 mm Hg. Interestingly, while increased osmolality decreased qtPA significantly at 140 mm Hg pCO2, it had no effect or even increased qtPA at 195 and 250 mm Hg. © 1996 John Wiley & Sons, Inc.