bldmutants of Streptomyces coelicolorA3(2) are blocked at the earliest stage of sporulation, the formation of aerial hyphae, and are pleiotropically defective in antibiotic production. Using a phage library of wild-type S. coelicolorDNA, we isolated a recombinant phage which restored both sporulation and antibiotic production to strains carrying the single known bldDmutation. Nucleotide sequence analysis of a 1.3-kb complementing subclone identified an open reading frame, designated bldD, encoding a translation product of 167 amino acid residues. Nucleotide sequence analysis of the bldD-containing fragment amplified from the chromosome of a bldDmutant strain revealed a point mutation changing a tyrosine residue at amino acid position 62 to a cysteine. Although a comparison of the BldD sequence to known proteins in the databases failed to show any strong similarities, analysis of the BldD sequence for secondary structural elements did reveal a putative helix-turn-helix, DNA recognition element near the C terminus of the protein. A comparison of bldDtranscript levels in the bldD+ and bldDmutant strains using both Northern blot analysis and S1 nuclease protection studies showed vast overexpression of bldDtranscripts in the mutant, suggesting that BldD negatively regulates its own synthesis. High-resolution S1 nuclease mapping identified the transcription start point as a G residue 63 nucleotides upstream from the bldDstart codon and 7 nucleotides downstream from −10 and −35 sequences resembling E. coli-like streptomycete promoters.