Aquatic microalgae induce a carbon-concentrating mechanism (CCM) to maintain photosynthetic activity in low-CO2 (LC) conditions. Although the molecular mechanism of the CCM has been investigated using the single-cell green alga Chlamydomonas reinhardtii, and several CCM-related genes have been identified by analyzing high-CO2 (HC)-requiring mutants, many aspects of the CO2-signal transduction pathways remain to be elucidated. In this study, we report the isolation of novel HC-requiring mutants defective in the induction of CCM by DNA tagging. Growth rates of 20,000 transformants grown under HC and LC conditions were compared, and three HC-requiring mutants (H24, H82, and P103) were isolated. The photosynthetic CO2-exchange activities of these mutants were significantly decreased compared with that of wild-type cells, and accumulation of HLA3 and both LCIA and HLA3 were absent in mutants H24 and H82, respectively. Although the insertion of the marker gene and the HC-requiring phenotype were linked in the tetrad progeny of H82, and a calcium-sensing receptor CAS was disrupted by the insertion, exogenous expression of CAS alone could not complement the HC-requiring phenotype.