The processes of CO2 acquisition were characterized for the acid-tolerant, free-living chlorophyte alga, CPCC 508. rDNA data indicate an affiliation to the genus Coccomyxa, but distinct from other known members of the genus. The alga grows over a wide range of pH from 3.0 to 9.0. External carbonic anhydrase (CA) was detected in cells grown above pH 5, with the activity increasing marginally from pH 7 to 9, but most of the CA activity was internal. The capacity for HCO3− uptake of cells treated with the CA inhibitor acetazolamide (AZA), was investigated by comparing the calculated rate of uncatalyzed CO2 formation with the rate of photosynthesis. Active bicarbonate transport occurred in cells grown in media above pH 7.0. Monitoring CO2 uptake and O2 evolution by membrane-inlet mass spectrometry demonstrated that air-grown cells reduced the CO2 concentration in the medium to an equilibrium concentration of 15 μM, but AZA-treated cells caused a drop in extracellular CO2 concentration to a compensation concentration of 27 μM at pH 8.0. CO2-pulsing experiments with cells in the light indicated that the cells do not actively take up CO2. An internal pool of unfixed inorganic carbon was not detected at the CO2 compensation concentration, probably because of the lack of active CO2 uptake, but was detectable at times before compensation point was reached. These results indicate that this free-living Coccomyxa possesses a CO2-concentrating mechanism (CCM) due to an active bicarbonate-uptake system, unlike the Coccomyxa sp. occurring in symbiotic association with lichens.