Agrobacterium tumefaciens possesses two catalases, a bifunctional catalase-peroxidase, KatA and a homologue of a growth phase regulated monofunctional catalase, CatE. In stationary phase cultures and in cultures entering stationary phase, total catalase activity increased 2-fold while peroxidase activity declined. katA and catE were found to be independently regulated in a growth phase dependent manner. KatA levels were highest during exponential phase and declined as cells entered stationary phase, while CatE was detectable at early exponential phase and increased during stationary phase. Only small increases in H₂O₂ resistance levels were detected as cells entering stationary phase. The katA mutant was more sensitive to H₂O₂ than the parental strain during both exponential and stationary phase. Inactivation of catE alone did not significantly change the level of H₂O₂ resistance. However, the katA catE double mutant was more sensitive to H₂O₂ during both exponential and stationary phase than either of the single catalase mutants. The data indicated that KatA plays the primary role and CatE acts synergistically in protecting A. tumefaciens from H₂O₂ toxicity during all phases of growth. Catalase-peroxidase activity (KatA) was required for full H₂O₂ resistance. The expression patterns of the two catalases in A. tumefaciens reflect their physiological roles in the protection against H₂O₂ toxicity, which are different from other bacteria.