The DnrI protein, essential for the biosynthesis of daunorubicin in Streptomyces peucetius, was purified almost to homogeneity from dnrI expression strains of Escherichia coll and S. peucetius through several steps of chromatography. The proteins purified from both organisms had identical chromatographic and electrophoretic behaviour. Purified Histagged or native DnrI was used to conduct DNA-binding assays by gel mobility-shift analysis, and the results showed no significant difference in the DNA-binding activity of native or His-tagged proteins. DnrI binds specifically to DNA segments containing the intergenic regions separating the putative dnrG-dpsABCD and dpsEF operons, and the dnrC gene and dnrDKPSQ operon. DNase I footprinting assays indicated that the DNA-binding sites for DnrI extended from upstream of the -10 to -35 regions of the dnrG or dpsE promoters to include about 65 bp of the dnrG-dpsE intergenic region and about 80 bp of the dnrC-dnrD intergenic region. Both binding sites contain imperfect inverted repeat sequences of 6-10 bp with a 5'-TCGAG-3' consensus sequence that was present in 4 out of 10 other promoter regions in the cluster of daunorubicin biosynthesis genes.