A Bacillus subtilis library of Tn917::lacZ insertions was screened for mutants that were unable to grow in the presence of normally sublethal concentrations of hydrogen peroxide. The identification and subsequent analysis of one mutant strain, YB2003, which carried the mutation designated kat-19, revealed that this strain was deficient in the expression of a vegetative catalase. Regions of the chromosome both 5' and 3' to the site of the Tn917 insertion, as well as the gene without the insertion (kat-19+) were cloned. The presence of the functional kat-19+ gene on a high-copy plasmid restored catalase activity to the kat-19::Tn917 strain as well as to strains of B. subtilis that carried the katA 1 mutation. While the katA+ locus is believed to represent the structural gene for the vegetative catalase of B. subtilis [Loewen and Switala, J. Bacteriol. 169 (1987) 5848-5851], the sequence analysis of the cloned kat-19+ DNA fragments revealed an open reading frame that showed significant homology between the deduced amino acid sequence of this gene product and that of known eukaryotic catalases.