At least 17 kb of DNA from the large unisolatable Streptomyces coelicolor A3(2) plasmid SCP1 are concerned with methylenomycin biosynthesis. Mutational cloning analysis, using insert-directed integration of att site deleted phage vectors into an SCP1-containing host, provided evidence of two large transcription units, of at least 6.6 kb and 9.5 kb. At the leftmost apparent end of the larger (left-hand) transcription unit is a region apparently involved in negative regulation of methylenomycin biosynthesis: when fragments from this region were used to direct phage integration, marked overproduction of methylenomycin resulted. The methylenomycin resistance determinant is located at the rightmost end of this same transcription unit. Hybridisation analysis with 13 kb of the cloned mmy region showed that it was closely similar to a segment of pSV1, a plasmid that specifies methylenomycin biosynthesis in S. violaceus-ruber SANK 95570.