The green alga Chlamydomonas reinhardtii possesses a CO2 concentratingmechanism (CCM) which helps in successful acclimationto low CO2 conditions. Current models of the CCM postulate that aseries of ion transporters bring HCO3- from outside the cell to thethylakoid lumen, where the carbonic anhydrase CAH3 dehydratesaccumulated HCO3- to CO2, raising the CO2 concentration forRubisco. Previously, HCO3- transporters have been identified atboth the plasma membrane and the chloroplast envelope, butthe transporter thought to be on the thylakoid membrane hasnot been identified. Three paralogous genes (BST1, BST2, BST3)belonging to the bestrophin family have been found to be upregulatedin low CO2 conditions, and their expression is controlledby CIA5, a transcription factor that controls many CCM genes.YFP fusions demonstrate that all three proteins are located onthe thylakoid membrane, and interactome studies indicate thatthey might associate with chloroplast CCM components. A singlemutant defective in BST3 still grows nearly normally on low CO2,indicating that the three bestrophin-like proteins may have redundantfunctions. Therefore, an RNAi approach was adopted to reducethe expression of all three genes at once. RNAi mutants withreduced expression of BST1-3 were unable to grow at low CO2concentrations, exhibited a reduced affinity to inorganic carboncompared to the wild type cells, and showed reduced inorganiccarbon uptake. We propose that these bestrophin-like proteins areessential components of the CCM that deliver HCO3- accumulatedin the chloroplast stroma to CAH3 inside the thylakoid lumen.