<jats:title>ABSTRACT</jats:title> <jats:p> Clavulanic acid is a potent inhibitor of β-lactamase enzymes and is of demonstrated value in the treatment of infections by β-lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strain <jats:italic>Streptomyces clavuligerus</jats:italic> were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219–236, <jats:italic>In</jats:italic> V. P. Gullo et al., ed., <jats:italic>Development in industrial microbiology series</jats:italic> , 1993). In contrast, we report the identification of three new genes, <jats:italic>orf10</jats:italic> ( <jats:italic>cyp</jats:italic> ), <jats:italic>orf11</jats:italic> ( <jats:italic>fd</jats:italic> ), and <jats:italic>orf12</jats:italic> , that are required for clavulanic acid biosynthesis as indicated by gene replacement and <jats:italic>trans</jats:italic> -complementation analysis in <jats:italic>S. clavuligerus</jats:italic> . These genes are contained within a 3.4-kb DNA fragment located directly downstream of <jats:italic>orf9</jats:italic> ( <jats:italic>cad</jats:italic> ) in the clavulanic acid cluster. While the <jats:italic>orf10</jats:italic> ( <jats:italic>cyp</jats:italic> ) and <jats:italic>orf11</jats:italic> ( <jats:italic>fd</jats:italic> ) proteins show homologies to other known <jats:italic>CYP-150</jats:italic> cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by <jats:italic>orf12</jats:italic> shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed.