Physiological properties of photosynthesis were determined in the marine diatom, Phaeodactylum tricornutum UTEX640, during acclimation from 5% CO2 to air and related to H2CO3 dissociation kinetics and equilibria in artificial seawater. The concentration of dissolved inorganic carbon at half maximum rate of photosynthesis (K0·5[DIC]) value in high CO2-grown cells was 1009 mmol m−3 but was reduced three-fold by the addition of bovine carbonic anhydrase (CA), whereas in air-grown cells K0·5[DIC] was 71 mmol m−3, irrespective of the presence of CA. The maximum rate of photosynthesis (Pmax) values varied between 300 and 500 μmol O2 mg Chl−1 h−1 regardless of growth pCO2. Bicarbonate dehydration kinetics in artificial seawater were re-examined to evaluate the direct HCO3− uptake as a substrate for photosynthesis. The uncatalysed CO2 formation rate in artificial seawater of 31·65°/oo of salinity at pH 8·2 and 25 °C was found to be 0·6 mmol m−3 min−1 at 100 mmol m−3 DIC, which is 53·5 and 7·3 times slower than the rates of photosynthesis exhibited in air- and high CO2-grown cells, respectively. These data indicate that even high CO2-grown cells of P. tricornutum can take up both CO2 and HCO3− as substrates for photosynthesis and HCO3− use improves dramatically when the cells are grown in air. Detailed time courses were obtained of changes in affinity for DIC during the acclimation of high CO2-grown cells to air. The development of high-affinity photosynthesis started after a 2–5 h lag period, followed by a steady increase over the next 15 h. This acclimation time course is the slowest to be described so far. High CO2-grown cells were transferred to controlled DIC conditions, at which the concentrations of each DIC species could be defined, and were allowed to acclimate for more than 36 h. The K0·5[DIC] values in acclimated cells appeared to be correlated only with [CO2(aq)] in the medium but not to HCO3−, CO32−, total [DIC] or the pH of the medium and indicate that the critical signal regulating the affinity of cells for DIC in the marine diatom, P. tricornutum, is [CO2(aq)] in the medium.