Two freshwater macrophytes, Ottelia alismoides and O. acuminata, were grown at low (mean5 lmol L-1) and high (mean 400 lmol L-1) CO2 concentrations under natural conditions. The ratio of PEPC to RuBisCO activity was 1.8 in O. acuminata in both treatments. In O. alismoides, this ratio was 2.8 and 5.9 when grown at high and low CO2, respectively, as a result of a twofold increase in PEPC activity. The activity of PPDK was similar to, and changed with, PEPC (1.9-fold change). The activity of the decarboxylating NADP-malic enzyme (ME) was very low in both species, while NAD-ME activity was high and increased with PEPC activity in O. alismoides. These results suggest that O. alismoides might perform a type of C4 metabolism with NAD-ME decarboxylation, despite lacking Kranz anatomy. The C4-activity was still present at high CO2 suggesting that it could be constitutive. O. alismoides at low CO2 showed diel acidity variation of up to 34 lequiv g-1 FW indicating that it may also operate a form of crassulacean acid metabolism(CAM). pH-drift experiments showed that both species were able to use bicarbonate. In O. acuminata, the kinetics of carbon uptake were altered by CO2 growth conditions, unlike in O. alismoides. Thus, the two species appear to regulate their carbon concentrating mechanisms differently in response to changing CO2. O. alismoides is potentially using three different concentrating mechanisms. The Hydrocharitaceae have many species with evidence for C4, CAM or some other metabolism involving organic acids, and are worthy of further study.