The effect of increasing levels of proclavaminate amidino hydrolase (Pah) on the rate of clavulanic acid production in Streptomyces clavuligerus ATCC 27064 was evaluated by increasing dosoge of a gene (pah2) encoding Pah. A strain (SMF5703) harboring a multicopy plasmid containing the pah2 gene showed significantly retarded cell growth and reduced clavulanic acid production, possibly attributable to the deleterious effects of the multicopy plasmid. In contrast, a strain (SMF5704) carrying a single additional copy of pah2 introduced into chromosome via an integrative plasmid showed enhanced production of clavulanic acid and increased levels of pah2 transcripts. Analysis of transcripts of other genes involved in the clavulanic acid biosynthetic pathway revealed a pattern similar to that seen in the parent. From these results, it appears that clavulanic acid production can be enhanced by duplication of pah2 through integration of a second copy of the gene into chromosome. However, increasing the copy number of only one gene, such as pah2, does not affect the expression of other pathway genes, and so only modest improvements in clavulanic acid production can be expected. Flux controlled by Pah did increase when the copy number of pah2 was doubled, suggesting that under these growth conditions, Pah levels may be a limiting factor regulating the rate of clavulanic acid biosynthesis in S. clavuligerus.