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 CO, and bicarbonate, increased pC0, is associated with increased osmolality in bioreactors with pH control. While a few preliminary reports indicate that elevated pC0, levels can inhibit cell growth and/or recombinant protein production, no comprehensive analysis of the interrelated effects of elevated pC0, and osmolality has been published. We have examined the effects of 140, 195, and 250 m m Hg (187, 260, and 333 mbar, respectively) pC0, (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 pC0, of 36 mm Hg. Elevated pC0, at 310 mOsm/kg osmolality inhibited cell growth in a dose-dependent fashion, with a maximum decrease of 30% in the specific growth rate ( p ) at 250 m m Hg. Osmolality alone had no effect on p, but the combination of elevated pCOz and osmolality increased the maximal reduction in p to 45%. Elevated pC0, at 310 mOsm/kg osmolality decreased the specific tPA production rate (9tpA) by up to 40% at 250 m m Hg. Interestingly, while increased osmolality decreased 9tpA significantly at 140 m m Hg pCO,, it had no effect or even increased 9tpA at 195 and 250 mm Hg.