We have determined the nucleotide sequence of a 2375-base-pair DNA fragment, which contained the sporulation gene spo0A cloned from Bacillus subtilis. The sequence had only one long open reading frame consisting of 239 codons, which was found to correspond to the spo0A gene by comparing the nucleotide sequence of the wild-type gene with those of the mutant alleles. The calculated molecular weight of the product of the wild-type spo0A gene was 26,500. We found also a new mutation, sgi, which maps within the spo0A gene. This mutation relieves the growth inhibition of the host cells caused by a multicopy plasmid carrying the spo0A gene. The mutations spo0A12, spo0C9V, and sgi-1 were found to be an amber mutation at the 62nd codon, a missense at the 229th codon, and a frame-shift at the 223rd codon of the spo0A gene, respectively.