<jats:title>ABSTRACT</jats:title> <jats:p> In the present study, we successfully cloned a 21-kb DNA fragment containing a <jats:sc>d</jats:sc> -cycloserine (DCS) biosynthetic gene cluster from a DCS-producing <jats:italic>Streptomyces</jats:italic> <jats:italic>lavendulae</jats:italic> strain, ATCC 11924. The putative gene cluster consists of 10 open reading frames (ORFs), designated <jats:italic>dcsA</jats:italic> to <jats:italic>dcsJ</jats:italic> . This cluster includes two ORFs encoding <jats:sc>d</jats:sc> -alanyl- <jats:sc>d</jats:sc> -alanine ligase ( <jats:italic>dcsI</jats:italic> ) and a putative membrane protein ( <jats:italic>dcsJ</jats:italic> ) as the self-resistance determinants of the producer organism, indicated by our previous work. When the 10 ORFs were introduced into DCS-nonproducing <jats:italic>Streptomyces lividans</jats:italic> 66 as a heterologous host cell, the transformant acquired DCS productivity. This reveals that the introduced genes are responsible for the biosynthesis of DCS. As anticipated, the disruption of <jats:italic>dcsG</jats:italic> , seen in the DCS biosynthetic gene cluster, made it possible for the strain ATCC 11924 to lose its DCS production. We here propose the DCS biosynthetic pathway. First, <jats:sc>l</jats:sc> -serine is O acetylated by a <jats:italic>dcsE-</jats:italic> encoded enzyme homologous to homoserine <jats:italic>O</jats:italic> -acetyltransferase. Second, <jats:italic>O</jats:italic> -acetyl- <jats:sc>l</jats:sc> -serine accepts hydroxyurea <jats:italic>via</jats:italic> an <jats:italic>O</jats:italic> -acetylserine sulfhydrylase homolog ( <jats:italic>dcsD</jats:italic> product) and forms <jats:italic>O</jats:italic> -ureido- <jats:sc>l</jats:sc> -serine. The hydroxyurea must be supplied by the catalysis of a <jats:italic>dcsB</jats:italic> -encoded arginase homolog using the <jats:sc>l</jats:sc> -arginine derivative, <jats:italic>N</jats:italic> <jats:sup>G</jats:sup> -hydroxy- <jats:sc>l</jats:sc> -arginine. The resulting <jats:italic>O</jats:italic> -ureido- <jats:sc>l</jats:sc> -serine is then racemized to <jats:italic>O</jats:italic> -ureido- <jats:sc>d</jats:sc> -serine by a homolog of diaminopimelate epimerase. Finally, <jats:italic>O</jats:italic> -ureido- <jats:sc>d</jats:sc> -serine is cyclized to form DCS with the release of ammonia and carbon dioxide. The cyclization must be done by the <jats:italic>dcsG</jats:italic> or <jats:italic>dcsH</jats:italic> product, which belongs to the ATP-grasp fold family of protein.