Four regulated promoters that direct the transcription of genes (i.e., korA, tra, kilB, and korB) involved in the transfer of the Streptomyces plasmid pIJ101 were isolated following the in vitro fusion of plasmid DNA fragments to a promoterless gene encoding the S. lividans extracellular enzyme beta-galactosidase. Introduction of pIJ101 into cells carrying each of these promoter-lac fusions resulted in decreased lac expression. The sites of initiation of transcription by the promoter sequences were identified by primer extension experiments, and the DNA sequences specifically required for promoter activity and regulation by pIJ101-encoded functions were determined by deletion analysis. The data obtained indicate that the korB locus encodes a repressor that regulates its own transcription, as well as transcription of the kilB promoter; korA and tra are transcribed from overlapping divergent promoters that are coregulated by the korA gene product. Common DNA sequence domains within coregulated promoters allowed the identification of putative binding sites for each of the kor gene products.