<jats:title>ABSTRACT</jats:title> <jats:p> An approximately 12.5-kbp region of DNA sequence from beyond the end of the previously described clavulanic acid gene cluster was analyzed and found to encode nine possible open reading frames (ORFs). Involvement of these ORFs in clavulanic acid biosynthesis was assessed by creating mutants with defects in each of the ORFs <jats:italic>. orf12</jats:italic> and <jats:italic>orf14</jats:italic> had been previously reported to be involved in clavulanic acid biosynthesis. Now five additional ORFs are shown to play a role, since their mutation results in a significant decrease or total absence of clavulanic acid production. Most of these newly described ORFs encode proteins with little similarity to others in the databases, and so their roles in clavulanic acid biosynthesis are unclear. Mutation of two of the ORFs, <jats:italic>orf15</jats:italic> and <jats:italic>orf16</jats:italic> , results in the accumulation of a new metabolite, <jats:italic>N</jats:italic> -acetylglycylclavaminic acid, in place of clavulanic acid. <jats:italic>orf18</jats:italic> and <jats:italic>orf19</jats:italic> encode apparent penicillin binding proteins, and while mutations in these genes have minimal effects on clavulanic acid production, their normal roles as cell wall biosynthetic enzymes and as targets for β-lactam antibiotics, together with their clustered location, suggest that they are part of the clavulanic acid gene cluster.