Cultures ofStreptomyces coelicolorA3 (2) produced actinorhodin in defined media with various carbon and nitrogen sources. Production occurred during biomass accumulation if assimilation of either the carbon or the nitrogen source limited the rate of growth. High growth rates tended to delay product synthesis until after biomass accumulation was complete, but fully biphasic fermentation profiles were achieved only with media supporting very rapid growth. The onset of actinorhodin production then coincided with a decline in the growth rate during transition of carbon-sufficient cultures to stationary phase. In cultures with maltose as a growth-limiting carbon source, depletion of phosphate increased the rate of actinorhodin biosynthesis, but did not alter the timing of its initiation. With defined media, the use of spores rather than vegetative mycelium as inocula reduced the overlap between trophophase and idiophase. The general guidelines for achieving biphasic production of actinorhodin inS.coelicolorA3 (2) cultures could be used to obtain trophophase–idiophase separation in cultures ofStreptomyces venezuelaeproducing chloramphenicol. However, the conditions needed to be modified to give optimized biphasic fermentations with individual strains. Under conditions favouring chloramphenicol production in a distinct idiophase, aromatic amine secondary metabolites in the same cultures ofS.venezuelaewere produced in a pattern that overlapped the trophophase, suggesting that conditions need to be tailored also to meet differences in the regulation of secondary metabolites.Key words:Streptomyces coelicolorA3 (2),Streptomyces venezuelae, actinorhodin, biphasic fermentations, chloramphenicol, inoculum shift down.