<jats:title>ABSTRACT</jats:title> <jats:p> Portions of the <jats:italic>Streptomyces clavuligerus</jats:italic> chromosome flanking <jats:italic>cas1</jats:italic> , which encodes the clavaminate synthase 1 isoenzyme (CAS1), have been cloned and sequenced. Mutants of <jats:italic>S. clavuligerus</jats:italic> disrupted in <jats:italic>cvm1</jats:italic> , the open reading frame located immediately upstream of <jats:italic>cas1</jats:italic> , were constructed by a gene replacement procedure. Similar techniques were used to generate <jats:italic>S. clavuligerus</jats:italic> mutants carrying a deletion that encompassed portions of the two open reading frames, <jats:italic>cvm4</jats:italic> and <jats:italic>cvm5</jats:italic> , located directly downstream of <jats:italic>cas1</jats:italic> . Both classes of mutants still produced clavulanic acid and cephamycin C but lost the ability to synthesize the antipodal clavam metabolites clavam-2-carboxylate, 2-hydroxymethyl-clavam, and 2-alanylclavam. These results suggested that <jats:italic>cas1</jats:italic> is clustered with genes essential and specific for clavam metabolite biosynthesis. When a <jats:italic>cas1</jats:italic> mutant of <jats:italic>S. clavuligerus</jats:italic> was constructed by gene replacement, it produced lower levels of both clavulanic acid and most of the antipodal clavams except for 2-alanylclavam. However, a double mutant of <jats:italic>S. clavuligerus</jats:italic> disrupted in both <jats:italic>cas1</jats:italic> and <jats:italic>cas2</jats:italic> produced neither clavulanic acid nor any of the antipodal clavams, including 2-alanylclavam. This outcome was consistent with the contribution of both CAS1 and CAS2 to a common pool of clavaminic acid that is shunted toward clavulanic acid and clavam metabolite biosynthesis.