<jats:title>ABSTRACT</jats:title> <jats:p> Past genetic studies have indicated that the genes encoding early enzymes of clavulanic acid biosynthesis may be duplicated in <jats:italic>Streptomyces clavuligerus</jats:italic> . We observed cross-hybridizing bands upon Southern analyses of proclavaminate amidinohydrolase ( <jats:italic>pah</jats:italic> )-defective mutant strains of <jats:italic>S. clavuligerus</jats:italic> screened with a <jats:italic>pah</jats:italic> -specific probe. The DNA fragment responsible for this cross hybridization was cloned and sequenced and shown to encode a second copy of the <jats:italic>pah</jats:italic> gene. The new <jats:italic>pah</jats:italic> gene ( <jats:italic>pah1</jats:italic> ) was 1,056 bp in length, and its sequence was 72% identical to that of the original <jats:italic>pah</jats:italic> gene ( <jats:italic>pah2</jats:italic> ). Disruption mutants with defects in <jats:italic>pah1</jats:italic> showed no significant effects on production of clavulanic acid or any of the clavam metabolites with stereochemistries opposite that of clavulanic acid ( <jats:italic>5S</jats:italic> clavams) produced by <jats:italic>S. clavuligerus</jats:italic> when they were grown on starch asparagine or soy medium. However, double mutants with defects in both <jats:italic>pah1</jats:italic> and <jats:italic>pah2</jats:italic> were defective in the production of both clavulanic acid and all of the <jats:italic>5S</jats:italic> clavam metabolites.