The culture levels of glucose and CO2 have been reported to independently have important influences on mammalian cell processes. In this work the combined effects of glucose limitation and CO2 partial pressure (pCO2) on monoclonal antibody (IgG) producing Chinese Hamster Ovary cells were investigated in a perfusion reactor operated with controlled cell specific medium feed rate, pH and osmolality. Under high glucose conditions (14.3 ± 0.8 mM), the apparent growth rate decreased (from 0.021 to 0.009 h−1) as the pCO2 increased to ∼220 mmHg, while the cell specific IgG productivity was almost unchanged. The lactate yield from glucose was not affected by pCO2 up to ∼220 mmHg and glucose was mainly converted to lactate. A feed medium modification from high (33 mM) to low (6 mM) glucose resulted in <0.1 mM glucose in the culture. As a result of apparently shifting metabolism towards the conversion of pyruvate to CO2, both the ratio of lactate to glucose and the alanine production rate were lowered (1.51–1.14 and 17.7–0.56 nmol/106 cells h, respectively). Interestingly, when the pCO2 was increased to ∼140 mmHg, limiting glucose resulted in 1.7-fold higher growth rates, compared to high glucose conditions. However, at ∼220 mmHg pCO2 this beneficial effect of glucose limitation on these CHO cells was lost as the growth rate dropped dramatically to 0.008 h−1 and the IgG productivity was lowered by 15% (P < 0.01) relative to the high glucose condition. The IgG galactosylation increased under glucose- limited compared to high-glucose conditions Biotechnol. Bioeng. 2007;97:1479–1488. © 2007 Wiley Periodicals, Inc.