A putative sigma factor gene, orf21, was disrupted or overexpressed in the wild-type clavulanic acid (CA) producer Streptomyces clavuligerus NRRL3585 and characterized. An orf21 mutant (Streptomyces clavuligerus HN14) of S. clavuligerus was obtained by insertional inactivation via double-crossover. Although there was little reduction of sporulation in the mutant, the growth pattern was similar between mutant and wild-type. The production was reduced by 10-15% in S. clavuligerus HN14 compared to that in wild-type. Overexpression of orf21 in wild-type cells caused hyperproduction of spores on solid medium and increased clavulanic acid production by 1.43-fold. The overexpression of orf21 in wild-type S. clavuligerus stimulated the expression of the early clavulanic acid genes, ceas2 and cas2, and the regulatory gene, ccaR, as demonstrated by RT-PCR. The elevation of the ceas2, cas2 and ccaR transcripts was consistent with the enhanced production of clavulanic acid.