<jats:title>ABSTRACT</jats:title> <jats:p> C-1027, the most potent member of the enediyne antitumor antibiotic family, is produced by <jats:italic>Streptomyces globisporus</jats:italic> C-1027 and consists of an apoprotein (encoded by the <jats:italic>cagA</jats:italic> gene) and a nonpeptidic chromophore. The C-1027 chromophore could be viewed as being derived biosynthetically from a benzoxazolinate, a deoxyamino hexose, a β-amino acid, and an enediyne core. By adopting a strategy for cloning of the C-1027 biosynthesis gene cluster by mapping a putative dNDP-glucose 4,6-dehydratase (NGDH) gene to <jats:italic>cagA</jats:italic> , we have localized 75 kb of contiguous DNA from <jats:italic>S. globisporus</jats:italic> . DNA sequence analysis of two regions of the cloned gene cluster revealed two genes, <jats:italic>sgcA</jats:italic> and <jats:italic>sgcB</jats:italic> , that encode an NGDH enzyme and a transmembrane efflux protein, respectively, and confirmed that the <jats:italic>cagA</jats:italic> gene resides approximately 14 kb upstream of the <jats:italic>sgcAB</jats:italic> locus. The involvement of the cloned gene cluster in C-1027 biosynthesis was demonstrated by disrupting the <jats:italic>sgcA</jats:italic> gene to generate C-1027-nonproducing mutants and by complementing the <jats:italic>sgcA</jats:italic> mutants in vivo to restore C-1027 production. These results represent the first cloning of a gene cluster for enediyne antitumor antibiotic biosynthesis and provide a starting point for future genetic and biochemical investigations of C-1027 biosynthesis.