<jats:title>Summary</jats:title><jats:p>RT‐PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the <jats:italic>ceaS2–bls2–pah2–cas2</jats:italic>, <jats:italic>cyp–fd–orf12–orf13</jats:italic> and <jats:italic>oppA2–orf16</jats:italic> genes, whereas <jats:italic>oat2</jats:italic>, <jats:italic>car, oppA1</jats:italic>, <jats:italic>claR</jats:italic>, <jats:italic>orf14</jats:italic>, <jats:italic>gcaS</jats:italic> and <jats:italic>pbpA</jats:italic> were expressed as monocistronic transcripts. Quantitative RT‐PCR of <jats:italic>Streptomyces clavuligerus</jats:italic> ATCC 27064 and the mutant <jats:italic>S. clavuligerus ccaR::aph</jats:italic> showed that, in the mutant, there was a 1000‐ to 10 000‐fold lower transcript level for the <jats:italic>ceaS2</jats:italic> to <jats:italic>cas2</jats:italic> polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the <jats:italic>ccaR</jats:italic> mutant for other genes in the cluster. Two‐dimensional electrophoresis and MALDI‐TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r‐CcaR protein showed that it bound to the <jats:italic>ceaS2</jats:italic> and <jats:italic>claR</jats:italic> promoter regions in the clavulanic acid cluster, and to the <jats:italic>lat</jats:italic>, <jats:italic>cefF</jats:italic>, <jats:italic>cefD–cmcI</jats:italic> and <jats:italic>ccaR</jats:italic> promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r‐CcaR, and allowed identification of heptameric sequences as CcaR binding sites.