The external inorganic carbon pool (CO(2) + HCO(3) (-)) was measured in both high and low CO(2)-grown cells of Chlamydomonas reinhardtii, using a silicone oil layer centrifugal filtering technique. The average internal pH values were measured for each cell type using [(14)C]dimethyloxazolidinedione, and the internal inorganic carbon pools were recalculated on a free CO(2) basis. These measurements indicated that low CO(2)-grown cells were able to concentrate CO(2) up to 40-fold in relation to the external medium. Low and high CO(2)-grown cells differed in their photosynthetic affinity for external CO(2). These differences could be most readily explained as being due to the relative CO(2)-concentrating capacity of each cell type. This physiological adaptation appeared to be based on changes in the abilities of the cells actively to accumulate inorganic carbon using an energy-dependent transport system.The energy dependence of CO(2) accumulation was investigated, using the inhibitors methyl viologen, 3-(3,4-dichlorophenyl)-1,1 dimethylurea, carbonyl cyanide trifluoromethoxyphenylhydrazone, and 3,5-di-tert-butyl-4-hydroxybenzylide nemalononitrile. It appears that the concentrating mechanism in both cell types may be dependent upon an energy supply linked to both phosphorylation in general and photophosphorylation. The treatment of low CO(2)-grown cells with the carbonic anhydrase inhibitor ethoxyzolamide decreased the apparent photosynthetic affinity for CO(2). This was correlated with a decrease in the transport of inorganic carbon into the cells.The nature of the CO(2)-concentrating mechanism, particularly with respect to a bicarbonate transport system, is discussed, and its possible occurrence in other algae is assessed.