Abstract: Selection of microalgae strains that are capable of utilizing high concentration of CO₂ is essential for industrial application. At present, the microalgae research mainly focus on the biomass, bioactive products and recovery properties, rather than physiological properties. The influence of different CO₂ concentrations, including air, 5% CO₂ and 20% CO₂ (v/v), on the growth and physiological properties of Chlorella sp. FACHB-1580 and Scenedesmus sp. FACHB-1618 were studied. The goal was to clarify the relationship between the physiological properties and the CO₂ utilizing capacity. Results showed that both strains were capable to use high concentration of CO₂ and had the maximum biomass accumulation, specific growth rate and CO₂ fixation rate under 5% CO₂ (v/v) culture condition. The maximum biomass was 3.5 g/L in Chlorella sp. FACHB-1580 and 5.4 g/L in Scenedesmus sp. FACHB-1618 which was 1.41 and 1.46 times higher in comparison with the control (air) group, respectively. Both strains had higher biomass under 20% CO₂ (v/v) condition than that in the air group (P < 0.05). With the increasing supply of CO₂, the affinity of inorganic carbon, the activity of intracellular (CA_int) and extracellular (CA_ext) carbonic anhydrase, as well as the initial activity and activation rate of Rubisco declined in both strains. However, there was no significant difference in the total Rubisco activity across time. In addition, Chlorella sp. FACHB-1580 had significantly higher levels of CA_int and CA_ext activities. By contrast, Scenedesmus sp. FACHB-1618 had very poor CA_int and CA_ext activities and were not detectable, indicating these two strains differed significantly in the utility of inorganic carbon. It was speculated that Chlorella sp. FACHB-1580 can absorb both CO₂ and \rmHCO_3^ - whereas the Scenedesmus sp. FACHB-1618 tends to only absorb CO₂. Chlorella sp. FACHB-1580 may obtain its abundant CO₂ for Rubisco via high CA_int activity; for Scenedesmus sp. FACHB-1618, due to its lower CA_int activity, it may increase the amount of CA protein, or higher carboxylase affinity for CO₂ to promote the reactions of photosynthesis.