Expression of the gene (daao) encoding D-amino acid oxidase (DAAO) in Escherichia coli typically results in a marked decrease of cell viability, and it has generally been assumed that the consumption of intracellular D-alanine by DAAO is responsible for this effect. Vitreoscilla hemoglobin (VHb) gene (vgb) was coexpressed with Rhodosporidium toruloides D-amino acid oxidase in E. coli BL21(DE3) and BL21(DE3)pLysS, expression hosts differing in the stringency of suppressing basal transcription. Not only was the toxic effect of DAAO on cell growth relieved but also the pronounced cell lysis of BL21(DE3)pLysS caused by the expression of DAAO was prevented by coexpressing VHb with DAAO. As a result of the higher cell density achieved, DAAO activity about 1.5-fold higher than that of DAAO-expressing control strains could be obtained by DAAO/VHb-coexpressing strains. The relieving effect of VHb on DAAO toxicity resulted from its oxygen-binding ability. The low availability of free intracellular oxygen reduced DAAO activity and consequently its toxicity.