Transcriptional analysis of the ermE gene of Saccharopolyspora erythraea, which confers resistance to erythromycin by N6-dimethylation of 23S rRNA and which is expressed from two promoters, ermEp1 and ermEp2, revealed a complex regulatory region in which transcription is initiated in a divergent and overlapping manner. Two promoters (eryC1p1 and eryC1p2) were identified for the divergently transcribed erythromycin biosynthetic gene eryC1, which plays a role in the formation of desosamine or its attachment to the macrolide ring. Transcription from eryC1p2 starts at the same position as that of ermEp1, but on the opposite strand of the DNA helix, suggesting co-ordinate regulation of genes for erythromycin production and resistance. ermEp1 initiates transcription at, and one nucleotide before, the ermE translational start codon. Site-directed and deletion mutagenesis, combined with immunochemical analysis, demonstrated that the ermEp1 transcript is translated in the absence of a conventional ribosome-binding site to give rise to the full-length 23S rRNA methylase. Deletion of the -35 region of ermEp1 reduced, but did not abolish, promoter activity, reminiscent of the 'extended -10' class of bacterial promoters which, like ermEp1, possess TGN motifs immediately upstream of their -10 regions and which initiate transcription seven nucleotides downstream of the -10 region.