Macrophyte productivity supports the littoral food web in fresh waters where widespread active CO2 concentrating mechanisms (CCMs) allow their productivity to be maintained despite potential inorganic carbon limitation. We studied HCO3- acquisition, the most common CCM in macrophytes, in the freshwater monocot Ottelia alismoides and showed that the external carbonic anhydrase (CA) inhibitor acetazolamide (AZ) decreases the affinity for CO2 uptake and prevents HCO3- use. The anion exchanger (AE)/solute carrier (SLC) type HCO3- transporters inhibitor 4,4’-diisothio-cyanatostilbene-2,2’-disulfonate (DIDS), has a smaller effect on CO2 uptake but also prevents HCO3- use. Analysis of transcripts showed that putative αCA-1 and SLC4 HCO3- transporters are unaffected by acclimation of leaves to different CO2, in agreement with physiological measurements showing a constitutive HCO3- use. Therefore, it is likely that αCA-1 and SLC4 HCO3- transporters are the targets of AZ and DIDS, respectively. Altogether, these results are consistent with acquisition of HCO3- based on co-diffusion of CO2 and HCO3- through the boundary layer, conversion of HCO3- to CO2 at the plasmalemma by αCA-1 and in addition, transport of HCO3- across the plasmalemma by SLC4 transporters. A model of these processes has been produced that can be used to test inorganic carbon uptake in future experiments.