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Production of the polyketide antibiotic actinorhodin by Streptomyces coelicolor A3(2) was investigated using a defined medium with or without iron supplementation. Iron limitation was found to enhance the intracellular production and export of the pigmented antibiotic. The effect of iron deficiency was particularly pronounced when the bacterium was grown with nitrate instead of ammonium. Analysis of the excreted pigment led to the identification of the lactone form of actinorhodin, γ-actinorhodin.