Using mass-spectrometric measurements of 18O exchange from 13C18O2 we determined the activity of carbonic anhydrase (CA; EC 4.2.1.1) in chloroplast envelope membranes isolated from Chlamydomonas reinhardtii cw-15. Our results show an enrichment of CA activity in these fractions relative to the activity in the crude chloroplast. The envelope CA activity increased about 8-fold during the acclimation to low-CO2 conditions and was completely induced within the first 4 h after the transfer to air levels of CO2. The CA-activity was not dissociated from envelope membranes after salt treatment. In addition, no cross-reactivity with other CA isoenzymes of Chlamydomonas was observed in our chloroplast envelope membranes. All these observations indicated that the protein responsible for this activity was a new CA isoenzyme, which was an integral component of the chloroplast envelopes from Chlamydomonas. The catalytic properties of the envelope CA activity were completely different from those of the thylakoid isoenzyme, showing a high requirement for Mg2+ and a high sensitivity to ethoxyzolamide. Analysis of the integral envelope proteins showed that there were no detectable differences between high- and low-inorganic carbon (Ci) cells, suggesting that the new CA activity was constitutively expressed in both high- and low-Ci cells. Two different high-Ci-requiring mutants of C. reinhardtii, cia-3 and pmp-1, had a reduced envelope CA activity. We propose that this activity could play a role in the uptake of inorganic carbon at the chloroplast envelope membranes.