Carbon dioxide (CO(2)) and its hydration product bicarbonate (HCO(3)(-)) are essential molecules in various physiological processes of all living organisms. The reversible interconversion between CO(2) and HCO(3)(-) is in equilibrium. This reaction is slow without catalyst, but can be rapidly facilitated by Zn(2+)-metalloenzymes named carbonic anhydrases (CAs). To gain an insight into the function of multiple clades of fungal CA, we chose to investigate the filamentous fungi Aspergillus fumigatus and A. nidulans. We identified four and two CAs in A. fumigatus and A. nidulans, respectively, named cafA-D and canA-B. The cafA and cafB genes are constitutively, strongly expressed whereas cafC and cafD genes are weakly expressed but CO(2)-inducible. Heterologous expression of the A. fumigatus cafB, and A. nidulans canA and canB genes completely rescued the high CO(2)-requiring phenotype of a Saccharomyces cerevisiae Deltance103 mutant. Only the DeltacafA DeltacafB and DeltacanB deletion mutants were unable to grow at 0.033% CO(2), of which growth defects can be restored by high CO(2). Defects in the CAs can affect Aspergilli conidiation. Furthermore, A. fumigatus DeltacafA, DeltacafB, DeltacafC, DeltacafD and DeltacafA DeltacafB mutant strains are fully virulent in a low-dose murine infection.