Carbonic anhydrase (CA) isozymes were identified and isolated from three strains of Phaeodactylum tricornutum [University of Texas Culture Collection (UTEX 640), North Eastern Pacific Culture Collection at the University of British Columbia B31 and Culture Collection of Algae and Protozoa 1052/1A]. External (CAext) and internal CA activity was detected by potentiometric assay of intact cells and cell homogenates of air and high CO2‐grown cells. CAext was detected only in UTEX 640 grown under CO2‐limited conditions and present in trace amounts in cells grown on high CO2. CA isozymes in cells extracts were separated by cellulose acetate electrophoresis and by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. All three strains had two CA bands in common, while UTEX 640 had a third, faster‐running band which was absent from extracts of high CO2‐grown cells and thus was the external isozyme. The internal CA isoforms of the UTEX 640 strain were shown to have molecular masses of 28 and 25 kDa, and the external 24 kDa. A fourth CAext isozyme with a molecular weight of 23.5 kDa was later detected using a polyclonal CA antibody. The CA isozymes were low‐CO2‐inducible proteins because Western blot analysis, using a polyclonal antibody, indicated that CA expression was repressed in high CO2‐grown cells. CA localization, using both immunofluorescence and immunogold techniques, with air‐grown cells indicated that the CAext was located in the periplasmic space and on the cell membrane, whereas in high CO2‐grown cells only internal CA was detected.