Cyanobacteria possess light-dependent CO2 uptake activity that results in the net hydration of CO2 to HCO3- and may involve a protein-mediated carbonic anhydrase (CA)-like activity. This process is vital for the survival of cyanobacteria and may be a contributing factor in the ecological success of this group of organisms. Here, via isolation of mutants of Synechococcus sp. PCC7942 that cannot grow under low-CO2 conditions, we have identified two novel genes, chpX and chpY, that are involved in light-dependent CO2 hydration and CO2 uptake reactions; co-inactivation of both these genes abolished both activities. The function and mechanism of the CO2 uptake systems supported by each chp gene product differs, with each associated with functionally distinct NAD(P)H dehydrogenase (NDH-1) complexes. The ChpX system has a low affinity for CO2 and is dependent on photosystem I cyclic electron transport, whereas the inducible ChpY system has a high affinity for CO2 and is dependent on linear electron transport. We believe that ChpX and ChpY are involved in a unique, net hydration of CO2 to HCO3-, that is coupled electron flow within the NDH-1 complex on the thylakoid membrane.