Deinococcus radiodurans, which is extremely resistant to ionizing radiation and oxidative stress, is known to have three catalases (DR1998, DRA0146, and DRA0259). In this study, to investigate the role of each catalase, we constructed catalase mutants (Delta dr1998, Delta drA0146, and Delta drA0259) of D. radiodurans. Of the three mutants, Delta dr1998 exhibited the greatest decrease in hydrogen peroxide (H2O2) resistance and the highest increase in intracellular reactive oxygen species (ROS) levels following H2O2 treatments, whereas Delta drA0146 showed no change in its H2O2 resistance or ROS level. Catalase activity was not attenuated in Delta drA0146, and none of the three bands detected in an in-gel catalase activity assay disappeared in Delta drA0146. The purified His-tagged recombinant DRA0146 did not show catalase activity. In addition, the phylogenetic analysis of the deinococcal catalases revealed that the DR1998-type catalase is common in the genus Deinococcus, but the DRA0146-type catalase was found in only 4 of 23 Deinococcus species. Taken together, these results indicate that DR1998 plays a critical role in the anti-oxidative system of D. radiodurans by detoxifying H2O2, but DRA0146 does not have catalase activity and is not involved in the resistance to H2O2 stress. (C) 2015 Elsevier Inc. All rights reserved.