The prominence of insect cell culture has grown rapidly due to its ability to produce baculovirus biopesticides and recombinant proteins using the Baculovirus Expression Vector System. A critical problem in the mass production of these products is CO2 accumulation to inhibitory levels within the bioreactor. The current research investigated the effect of elevated CO2 concentrations on insect cell growth and metabolism and the roles of oxidative stress and intracellular pH (pHi) in CO2 inhibition. Spodoptera frugiperda Sf-9 insect cells were cultured in a 3 L bioreactor (1.2 L working volume) controlled at 20% air saturation, 27oC and a pH of 6.2. The cells were exposed to a constant CO2 concentration by purging the medium with CO2 and the headspace with air. The experiments were repeated for different CO2 concentrations and samples were taken every 24 h to determine cell density, viability, metabolism and oxidative stress. The population doubling time (PDT) of Sf-9 cells increased with increasing CO2 concentration. Specifically, the PDT for 0-37, 73, 147, 183 and 220 mm Hg CO2 concentrations were 23.2 ± 6.7, 32.4 ± 7.2, 38.1 ± 13.3, 42.9 ± 5.4 and 69.3 ± 35.9 h (n = 3 or 4; 95% confidence level), respectively. An 80 mL working volume shaker flask was maintained as a control and had an average PDT of 24.9 ± 3.1 h (n = 7; 95% confidence level). The viability of cells in all experiments was above 90%. The osmolality for all bioreactor experiments was observed to be 300 - 360 mOsm/kg, a range that is known to have a negligible effect on insect cell culture. Elevated CO2 concentration did not alter the cell specific glucose consumption rate (2.5 to 3.2 x 10-17 mol/cell-s), but slightly increased the specific lactate production rate from -3.0 x 10-19 mol/cell-s to 10.2 x 10-19mol/cell-s. Oxidative stress did not contribute to CO2 inhibition in uninfected Sf-9 cells as no significant increase in the levels of lipid hydroperoxide and protein carbonyl concentrations was discovered at elevated CO2 concentration. The experiments conducted to determine the effect of CO2 on pHi were not successful and different experimental methods tested were well documented.