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The effect of CO2 concentration on the rate of photorespiratory ammonium excretion and on glutamine synthetase (GS) and carbonic anhydrase (CA) isoenzymes activities has been studied in Chlamydomonas reinhardtii cw‐15 mutant (lacking cell wall) and in the high CO2‐requiring double mutant cia‐3/cw‐15 (lacking cell wall and chloroplastic carbonic anhydrase). In cw‐15 cells, both the extracellular (CAext) and chloroplastic (CAchl) CA activities increased after transferring cells from media bubbled with 5% CO2 in air (v/v, high‐Ci cells) to 0.03% CO2 (low‐Ci cells), whereas in cia‐3/cw‐15 cells only the CAext was induced after adaptation to low‐Ci conditions and the CAchl activity was negligible. During adaptation to low‐Ci conditions in the presence of 1 mM of l‐methionine‐D,L‐sulfoximine (MSX), a specific inhibitor of GS activity, both mutant strains excreted photorespiratory ammonium into nitrogen free medium. In addition, the ammonium excretion rate by cw‐15 in the presence of MSX was lower in cells grown and kept at 5% CO2 than in high‐Ci cells adapted to 0.03% CO2. The double mutant cia‐3/cw‐15 excreted photorespiratory ammonium at a higher rate than did cw‐15. Total GS activity (GS‐1 plus GS‐2) increased during adaptation to 0.03% CO2 in both strains of C. reinhardtii. However, only the activity GS‐2, which is located in the chloroplast, increased during the adaptation to low CO2, whereas the cytosolic GS‐1 levels remained similar in high and low‐Ci cells. We conclude that: (1) cia‐3/cw‐15 cells lack chloroplastic CA activity; (2) in C. reinhardtii photorespiratory ammonium is refixed in the chloroplasts through the GS‐2/GOGAT cycle; and (3) chloroplastic GS‐2 concentration changes in response to the variation of environmental CO2 concentration. [ABSTRACT FROM AUTHOR]