Bacterial β-class carbonic anhydrases (CAs) are zinc metalloenzymes catalysing reversible hydration of CO2. They maintain the intracellular balance of CO2/bicarbonate required for biosynthetic reactions and represent a new group of antimicrobial drug targets. Genome sequence analysis of Pseudomonas aeruginosa PAO1, an opportunistic human pathogen causing life threatening infections, identified three genes, PAO102, PA2053 and PA4676, encoding putative β-CAs that share 28-45 % amino acid sequence identity and belong to clades A and B. The genes are conserved among all sequenced pseudomonads. The CAs were cloned, heterologously expressed and purified. Metal and enzymic analyses confirmed that the proteins contain Zn(2+) and catalyse hydration of CO2 to bicarbonate. PAO102 (psCA1) was 19-26-fold more active, and together with PA2053 (psCA2) showed CA activity at both pH 7.5 and 8.3, whereas PA4676 (psCA3) was active only at pH 8.3. Circular dichroism spectroscopy suggested that psCA2 and psCA3 undergo pH-dependent structural changes. Taken together, the data suggest that psCA1 may belong to type I and psCA3 to type II β-CAs. Immunoblot analysis showed that all three CAs are expressed in PAO1 cells when grown in ambient air and at 5 % CO2; psCA1 appeared more abundant under both conditions. Growth studies of transposon mutants showed that the disruption of psCA1 impaired PAO1 growth in ambient air and caused a minor defect at high CO2. Thus, psCA1 contributes to the adaptation of P. aeruginosa to low CO2 conditions and will be further studied for its role in virulence and as a potential antimicrobial drug target in this organism.