DNA polymerase lambda (Pol lambda) is a DNA polymerase beta (Pol beta)-like enzyme with both DNA synthetic and 5'-deoxyribose-5'-phosphate lyase domains. Recent biochemical studies implicated Pol lambda as a backup enzyme to Pol beta in the mammalian base excision repair (BER) pathway. To examine the interrelationship between Pol lambda and Pol beta in BER of DNA damage in living cells, we disrupted the genes for both enzymes either singly or in combination in the chicken DT40 cell line and then characterized BER phenotypes. Disruption of the genes for both polymerases caused hypersensitivity to H(2)O(2)-induced cytotoxicity, whereas the effect of disruption of either polymerase alone was only modest. Similarly, BER capacity in cells after H(2)O(2) exposure was lower in Pol beta(-/-)/Pol lambda(-/-) cells than in Pol beta(-/-), wild-type, and Pol lambda(-/-) cells, which were equivalent. These results suggest that these polymerases can complement for one another in counteracting oxidative DNA damage. Similar results were obtained in assays for in vitro BER capacity using cell extracts. With MMS-induced cytotoxicity, there was no significant effect on either survival or BER capacity from Pol lambda gene disruption. A strong hypersensitivity and reduction in BER capacity was observed for Pol beta(-/-)/Pol lambda(-/-) and Pol beta(-/-) cells, suggesting that Pol beta had a dominant role in counteracting alkylation DNA damage in this cell system.