<jats:title>ABSTRACT</jats:title> <jats:p> Leinamycin (LNM), produced by <jats:italic>Streptomyces atroolivaceus</jats:italic> , is a thiazole-containing hybrid peptide-polyketide natural product structurally characterized with an unprecedented 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a 18-member macrolactam ring. LNM exhibits a broad spectrum of antimicrobial and antitumor activities, most significantly against tumors that are resistant to clinically important anticancer drugs, resulting from its DNA cleavage activity in the presence of a reducing agent. Using a PCR approach to clone a thiazole-forming nonribosomal peptide synthetase (NRPS) as a probe, we localized a 172-kb DNA region from <jats:italic>S. atroolivaceus</jats:italic> S-140 that harbors the <jats:italic>lnm</jats:italic> biosynthetic gene cluster. Sequence analysis of 11-kb DNA revealed three genes, <jats:italic>lnmG</jats:italic> , <jats:italic>lnmH</jats:italic> , and <jats:italic>lnmI</jats:italic> , and the deduced product of <jats:italic>lnmI</jats:italic> is characterized by domains characteristic to both NRPS and polyketide synthase (PKS). The involvement of the cloned gene cluster in LNM biosynthesis was confirmed by disrupting the <jats:italic>lnmI</jats:italic> gene to generate non-LNM-producing mutants and by characterizing LnmI as a hybrid NRPS-PKS megasynthetase, the NRPS module of which specifies for <jats:sc>l</jats:sc> -Cys and catalyzes thiazole formation. These results have now set the stage for full investigations of LNM biosynthesis and for generation of novel LNM analogs by combinatorial biosynthesis.