A physiological and molecular biological study was made of the control of methylenomycin biosynthesis by Streptomyces coelicolor A3(2). A simple and reliable assay for this antibiotic was developed. Conditions that permit the synthesis of methylenomycin by S. coelicolor cultures grown in defined medium were elucidated: a readily assimilated carbon and nitrogen source is required. Under these conditions methylenomycin is produced late in the growth phase, at the time of transition from exponential to linear growth. Provided that the phosphate concentration in the medium is kept high, there is synthesis of methylenomycin but not of the other secondary metabolites that this strain can produce. These conditions were used to study the transcription of the methylenomycin gene cluster during the transition from primary to secondary metabolism. The biosynthetic genes of at least one of the mmy transcription units appear to be transcribed before the mmr resistance determinant. The possibility that methylenomycin induces the transcription of mmr is discussed.