Many aquatic photosynthetic organisms exhibit a variety of acclimation responses to limiting CO2 availability, including induction of CO2 concentrating mechanisms (CCMs), which represent adaptations to environments with changing and, at times, limiting CO2 availability. Although the characteristics and regulation of limiting CO2 acclimation, including the CCM, in the green alga Chlamydomonas reinhardtii have been well demonstrated by physiological and biochemical studies, much less information is available at the molecular level with regard to the components involved in the CCM and other parts of the limiting CO2 acclimation response. We have applied insertional mutagenesis and genetic approaches in an effort to identify the components of the CCM, other pathways essential to limiting CO2 acclimation and of the regulatory pathways controlling limiting CO2 acclimation responses. Many mutants defective in acclimation to limiting CO2 environments were generated by gene tagging, and, although all the identified mutants are phenotypically high-CO2-requiring, they can be distinguished by differences in their ability to survive under various levels of CO2, by differences in heterotrophic growth, and by differences in the gene expression patterns of CO2 responsive genes. Using the insertional tags, several mutant genes have been identified or confirmed, including the transcription factor Cia5, 2-oxoglutarate dehydrogenase (Ogd2), heat shock proteins and LciB , which encodes an unknown protein apparently involved in inorganic carbon transport. The identity of defective genes in the mutants suggests that they are located in various metabolic or regulatory pathways, including the CCM, mitochondrial respiration, chloroplast photoprotection and some not yet identified regulatory pathways. In addition, it has become evident that analysis of insertional mutants in C. reinhardtii is rarely straightforward, even when there is a single insert that co-segregates with the mutant phenotype. We hope that this endeavor will continue to provide information useful in elucidating the networks associated with the limiting CO2 acclimation response and its regulation.