A 2.3-kilobase pair EcoRI fragment containing the spo0B locus has been sequenced. The spo0B locus was shown to code for a protein of 22,542 daltons. Promoter distal to the spo0B locus, an open reading frame was uncovered which was preceded by a strong ribosome-binding site. S1 nuclease protection experiments revealed that both the spo0B locus and this open reading frame were part of the same transcript. A portion of the middle of the open reading frame was cloned in the integrative vector pJH101. Transformation of this plasmid into Bacillus subtilis 168 was only rarely successful, and those few colonies that arose consisted of cells that had lost the plasmid. The results suggested that the product of this open reading frame is essential for the growth of the bacterium. The regulation of the spo0B locus was studied by using translational spo0B-lacZ fusions in an integrative vector. These studies revealed that the spo0B locus was maximally expressed during vegetative growth. It was estimated that 50 to 100 copies of the protein are present during this period. Sequence analysis of the region upstream from the spo0B locus revealed another operon that contained a gene coding for a protein homologous to ribosomal protein L27 of Escherichia coli.