Deoxyribonucleic acid (DNA)-binding proteins have been compared between wild-type Bacillus subtilis and five sporulation mutants blocked at different stage O loci. Extracts from exponentially growing cells have been fractionated for proteins binding to single-stranded calf thymus DNA-cellulose and double-stranded B. subtilis DNA-cellulose. In nutrient broth, stage O mutations cause an accumulation of proteins with affinity for double-stranded DNA. Suppression of the mutation with extragenic suppressors relieves the accumulation. In minimal glucose medium, the stage O mutations also cause accumulation of proteins with affinity for double-stranded DNA, but the species accumulated are different from those of nutrient broth-grown cells. In neither case did stage O mutations affect proteins with affinity for single-stranded DNA. The results suggest that the products of stage O loci are functional and operative during vegetative growth.