Carbonic anhydrases (CAs) are Zn-containing metalloenzymes that catalyse the reversible hydration of CO2. We investigated the αCA and βCA families in Arabidopsis, which contain eight αCA (AtαCA1-8) and six βCA genes (AtβCA1-6). Analyses of expressed sequence tags (ESTs) from The Arabidopsis Information Resource (TAIR) database indicate that all the βCA encoding sequences, but only three of the AtαCA, are expressed. Using semi-quantitative PCR experiments, functional CA genes were more strongly expressed in green tissue, but strong expression was also found in roots for βCA3, βCA6 and αCA2. Two αCA genes were shown to respond to the CO2 environment, while the others were unresponsive. Using the green fluorescent reporter protein gene fused with cDNA sequences coding for βCAs, we provided evidence that βCAs were targeted to specific subcellular compartments: βCA1 and βCA5 were targeted to the chloroplast, βCA2 and βCA3 to the cytosol, βCA4 to the plasma membrane and βCA6 to the mitochondria. The targeting and the pattern of gene expression suggest that CA isoforms play specific roles in subcellular compartments, tissues and organs. The data indicate that other CA isoforms than the well-characterized βCA1 may contribute to the CO2 transfer in the cell to the catalytic site of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco).