Hornworts (Anthocerotophyta) are the only group of land plants with pyrenoid-containing chloroplasts. CO2 exchange and carbon isotope discrimination values (Δ13C) values have previously demonstrated the presence of a CO2 concentrating mechanism (CCM) in some pyrenoid-containing species. We have examined hornwort CCM function by using a combined fluorometer/mass spectrometer based technique to compare pyrenoid-containing ( Phaeoceros Prosk. and Notothylas Sull.) and pyrenoid-lacking ( Megaceros Campbell) hornworts, with the liverwort Marchantia polymorpha L. that has standard C3 photosynthesis and a thalloid growth form similar to hornworts. We found that Notothylas has more CCM activity than Phaeoceros , and that Megaceros has the least CCM activity. Notothylas and Phaeoceros had compensation points from 11–13 parts per million (ppm) CO2, lower K 0.5(CO2) than Marchantia , negligible photorespiration, and they accumulate a pool of dissolved inorganic carbon (DIC) between 19–108 nmol mg–1 chlorophyll. Megaceros had an intermediate compensation point of 31 ppm CO2 (compared with 64 ppm CO2 in Marchantia ), a lower K 0.5(CO2) than Marchantia , and some photorespiration, but no DIC pool. We also determined the catalytic rate of carboxylation per active site of Rubisco for all four species ( Marchantia , 2.6 s–1; Megaceros , 3.3 s–1; Phaeoceros, 4.2 s–1; Notothylas 4.3 s-1), and found that Rubisco content was 3% of soluble protein for pyrenoid-containing species, 4% for Megaceros and 8% for Marchantia .