Carbonic anhydrase (CA) catalyzes the interconversion of CO2 and bicarbonate, the forms of inorganic carbon used by theprimary carboxylating enzymes of C3 and C4 plants, respectively. Multiple forms of CA are found in both photosyntheticsubtypes; however, the number of isoforms and the location and function of each have not been elucidated for any single plantspecies. Genomic Southern analyses showed that the C4 dicotyledon Flaveria bidentis ‘Kuntze’ contains a small gene familyencoding b-CA and cDNAs encoding three distinct b-CAs, named CA1, CA2, and CA3, were isolated. Quantitative reversetranscription-polymerase chain reactions showed that each member of this b-CA family has a specific expression pattern inF. bidentis leaves, roots, and flowers. CA3 transcripts were at least 50 times more abundant than CA2 or CA1 transcripts in leaves.CA2 transcripts were detected in all organs examined and were the most abundant CA transcripts in roots. CA1 mRNA levelswere similar to those of CA2 in leaves, but were considerably lower in roots and flowers. In vitro import assays showed CA1was imported into isolated pea (Pisum sativum) chloroplasts, whereas CA2 and CA3 were not. These results support thefollowing roles for F. bidentis CAs: CA3 is responsible for catalyzing the first step in the C4 pathway in the mesophyll cellcytosol; CA2 provides bicarbonate for anapleurotic reactions involving nonphotosynthetic forms of phosphoenolpyruvatecarboxylase in the cytosol of cells in both photosynthetic and nongreen tissues; and CA1 carries out nonphotosyntheticfunctions demonstrated by C3 chloroplastic b-CAs, including lipid biosynthesis and antioxidant activity.