The evolution of CO2 in a fed-batch culture of recombinant Escherichia coli containing human-like collagen (HLC) cDNA was determined with an O-2-enriched air supply (40%, v/v) in a 12.8 l fermentor; a maximum CO2 concentration of 12.7% in the effluent gas was detected. The CO2 pulse injection experiments showed that: (1) a 20% CO2 pulse introduced in the batch cultivation phases inhibited cell growth but if introduced in the fed-batch cultivation phases slightly stimulated growth; and (2) CO2 inhibited HLC expression only in the expression phase, where the final HLC concentration decreased by 34% under a 3 h 20% CO2 pulse. The higher the CO2 concentration and/or the longer the duration of the CO2 pulse, the stronger the stimulatory or inhibitory effects.