A 7023 nucleotide BamHI fragment immediately upstream of the eryK gene of the erythromycin (Er) biosynthesis cluster in Saccharopolyspora erythraea was sequenced. Computer-assisted analysis of this sequence reveals the existence of seven ORFs that display the codon preferences typical of actinomycete genes. Six of these show homology to known genes: an esterase, a transposase, a peptidyl-prolyl cis-trans isomerase, a subtilisin inhibitor-like protein, and two genes involved in bacterial cell wall biosynthesis. All the ORFs are transcribed toward the Er biosynthetic gene cluster (in the same direction as eryK). From the predicted functions of the putative ORF products none of these genes appear to be involved in the biosynthesis of Er. The eryK gene thus most likely defines one end of the Er biosynthetic gene cluster.