We determined the physical and transcriptional organisation of the set of previously cloned biosynthetic genes involved in the production of the polyketide antibiotic actinorhodin by Streptomyces coelicolor A3(2). Complementation and mutational cloning analyses (in part using new phi C31 phage vectors incorporating a transcriptional terminator to block transcription from vector promoters into the cloned DNA) indicate that all the biosynthetic genes, including at least one regulatory (activator) gene, are clustered in a chromosomal region of about 26 kb. The genes are organised in at least four separate transcription units, ranging in size from 1 kb for the class III gene, to a polycistronic transcript of at least 5 kb for the class I, VII and IV genes. Indirect evidence shows that resistance to actinorhodin is also determined by the cloned DNA.