<jats:title>ABSTRACT</jats:title> <jats:p> Recently, a second copy of a gene encoding proclavaminate amidinohydrolase ( <jats:italic>pah1</jats:italic> ), an enzyme involved in the early stages of clavulanic acid and clavam metabolite biosynthesis in <jats:italic>Streptomyces clavuligerus</jats:italic> , was identified and isolated. Using Southern analysis, we have now isolated second copies of the genes encoding the carboxyethylarginine synthase ( <jats:italic>ceaS</jats:italic> ) and β-lactam synthetase ( <jats:italic>bls</jats:italic> ) enzymes. These new paralogues are given the gene designations <jats:italic>ceaS1</jats:italic> and <jats:italic>bls1</jats:italic> and are located immediately upstream of <jats:italic>pah1</jats:italic> on the chromosome. Furthermore, sequence analysis of the region downstream of <jats:italic>pah1</jats:italic> revealed a second copy of a gene encoding ornithine acetyltransferase ( <jats:italic>oat1</jats:italic> ), thus indicating the presence of a cluster of paralogue genes. <jats:italic>ceaS1</jats:italic> , <jats:italic>bls1</jats:italic> , and <jats:italic>oat1</jats:italic> display 73, 60, and 63% identities, respectively, at the nucleotide level to the original <jats:italic>ceaS2</jats:italic> , <jats:italic>bls2</jats:italic> , and <jats:italic>oat2</jats:italic> genes from the clavulanic acid gene cluster. Single mutants defective in <jats:italic>ceaS1</jats:italic> , <jats:italic>bls1</jats:italic> , or <jats:italic>oat1</jats:italic> were prepared and characterized and were found to be affected to variable degrees in their ability to produce clavulanic acid and clavam metabolites. Double mutants defective in both copies of the genes were also prepared and tested. The <jats:italic>ceaS1</jats:italic> / <jats:italic>ceaS2</jats:italic> and the <jats:italic>bls1</jats:italic> / <jats:italic>bls2</jats:italic> mutant strains were completely blocked in clavulanic acid and clavam metabolite biosynthesis. On the other hand, <jats:italic>oat1</jats:italic> / <jats:italic>oat2</jats:italic> double mutants still produced some clavulanic acid and clavam metabolites. This may be attributed to the presence of the <jats:italic>argJ</jats:italic> gene in <jats:italic>S. clavuligerus</jats:italic> , which encodes yet another ornithine acetyltransferase enzyme that may be able to compensate for the lack of OAT1 and -2 in the double mutants.