The effects of pH, carbon dioxide vapor pressure, pCO(2), and temperature on El and E3 deleted recombinant adenovirus vector (rAV) production with HEK293S cells have been studied in the ranges of pH = 6.7-7.7, pCO(2) = 0.05-0.20 atm, and T = 32-39 degreesC, respectively. The experiments were performed in four 500-mL bioreactors in parallel, which make possible the reduction of inter-run variability. Cell concentration and viability, relative oxygen uptake rate (OUR), fluorescence, and viral titer were measured. It was found that, although pH and pCO(2) did not affect significantly cell viability in the range studied, they had an important effect on virus titer. pCO(2) allowed the maximum production of rAV at 0.05 atm, and pH showed a very sharp optimum at 7.2. Temperature had an effect on both cell metabolism and virus titer. Low temperature prolonged cell viability and high OUR. Most of all, a 3-fold increase in virus yield was found at 35degreesC compared to that at 37degreesC, while 32degreesC was not as beneficial (1.5-fold increase). This finding could have an important impact on large-scale production. This phenomenon was modeled using a simple 3-parameter synthesis-decay model. This model shows how the optimum gain in virus production at 35degreesC is due to a balance between the production and decay processes at that temperature.