The Propionibacterium freudenreichii hemB gene that encodes delta-aminolevulinic acid (ALA) dehydratase (EC 4.2.1.24), which catalyzes the synthesis of monopyrrole porphobilinogen, a precursor of tetrapyrrole biosynthesis, was cloned and characterized. A DNA fragment that could complement the hemB mutation of Escherichia coil was sequenced. An open reading frame (ORF) located downstream from a potential ribosome-binding site encoded a polypeptide of 332 amino acid residues, with a deduced molecular mass of 35,830 Da. The predicted amino acid sequence encoded by the ORF is similar to those of other ALA dehydratases. Two regions, the putative zinc-binding site and the predicted catalytic site including the invariant lysine residue of the active site, were highly conserved. The gene was confirmed to encode ALA dehydratase by determining the enzymatic activity of the transformant. After expression of the hemB gene, the N-terminal amino acid sequence of the gene product was identified with the deduced amino acid sequence, although the gene product moved more slowly than the expected molecular size of the monomer in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The M(r) was determined to be 290 k by gel filtration, The ALA dehydratase from P. freudenreichii is thus an octamer with a subunit of 36 kDa.