CO2 partial pressure (pCO(2)) in industrial cell culture reactors may reach 150-200 mm Hg, which can significantly inhibit cell growth and recombinant protein production. The inhibitory effects of elevated pCO(2) at constant pH are due to a combination of the increases in pCO(2) and [HCO3-], per se, and the associated increase in osmolality. To decouple the effects of pCO(2) and osmolality, low-salt basal media have been used to compensate for this associated increase in osmolality. Under control conditions (40 mm Hg-320 mOsm/kg), hybridoma cell growth and metabolism was similar in DMEM:F12 with 2% fetal bovine serum and serum-free HB GRO. In both media, pCO(2) and osmolality made dose-dependent contributions to the inhibition of hybridoma cell growth and synergized to more extensively inhibit growth when combined. Elevated osmolality was associated with increased apoptosis. In contrast, elevated pCO(2) did not increase apoptotic cell death. Specific antibody production also increased with osmolality although not with pCO(2). In an effort to understand the mechanisms through which elevated pCO(2) and osmolality affect hybridoma cells, glucose metabolism, glutamine metabolism, intracellular pH (pHi), and cell size were monitored in batch cultures. Elevated pCO(2) (with or without osmolality compensation) inhibited glycolysis in a dose-dependent fashion in both media. Osmolality had little effect on glycolysis. On the other hand, elevated pCO(2) alone had no effect on glutamine metabolism, whereas elevated osmolality increased glutamine uptake. Hybridoma mean pHi was similar to0.2 pH units lower than control at 140 mm Hg pCO(2) (with or without osmolality compensation) but further increases in pCO(2) did not further decrease pHi. Osmolality had little effect on pHi. Cell size was smaller than control at elevated pCO(2) at 320 mOsm/kg, and greater than control in hyperosmotic conditions at 40 mm Hg.