Bacillus subtilis contains two aa3-type terminal oxidases (caa3-605 and aa3-600) catalyzing cytochrome c and quinol oxidation, respectively, with the concomitant reduction of O2 to H2O (Lauraeus, M., Haltia, T., Saraste, M., and Wikström, M. (1991) Eur. J. Biochem. 197, 699-705). Previous studies characterized only the structural genes of caa3-605 oxidase. We isolated the genes coding for the four subunits of a B. subtilis terminal oxidase from a genomic DNA library. These genes, named qoxA to qoxD, are organized in an operon. Examination of the deduced amino acid sequence of Qox subunits showed that this oxidase is structurally related to the large family of mitochondrial-type aa3 terminal oxidases. In particular, the amino acid sequences are very similar to those of subunits of Escherichia coli bo quinol oxidase and B. subtilis caa3-605 cytochrome c oxidase. We produced, by in vitro mutagenesis, a mutation in the qox operon. From the phenotype of the mutant strain devoid of Qox protein, the study of expression of the qox operon in different growth conditions, and the analysis of the deduced amino acid sequence of the subunits, we concluded that Qox protein and aa3-600 quinol oxidase are the same protein. Although several terminal oxidases are found in B. subtilis, Qox oxidase (aa3-600) is predominant during the vegetative growth and its absence leads to important alterations of the phenotype of B. subtilis.