The occurrence of an active CO2 transport system and of carbonic anhydrase (CA) has been investigated by mass spectrometry in the marine, unicellular rhodophyte Porphyridium cruentum (S.F. Gray) Naegeli and two marine chlorophytes Nannochloris atomus Butcher and Nannochloris maculata Butcher. Illumination of darkened cells incubated with 100 μM H13CO3− caused a rapid initial drop, followed by a slower decline in the extracellular CO2 concentration. Addition of bovine CA to the medium raised the CO2 concentration by restoring the HCO3−–CO2 equilibrium, indicating that cells were taking up CO2 and were maintaining the CO2 concentration in the medium below its equilibrium value during photosynthesis. Darkening the cell suspensions caused a rapid increase in the extracellular CO2 concentration in all three species, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. CA activity was detected by monitoring the rate of exchange of 18O from 13C18O2 into water. Exchange of 18O was rapid in darkened cell suspensions, but was not inhibited by 500 μM acetazolamide, a membrane-impermeable inhibitor of CA, indicating that external CA activity was not present in any of these species. In all three species, the rate of exchange was completely inhibited by 500 μM ethoxyzolamide, a membrane-permeable CA-inhibitor, showing that an intracellular CA was present. These results demonstrate that the three species are capable of CO2 uptake by active transport for use as a carbon source for photosynthesis.