Addition of cerulenin (0.25-1.0 mM) to cultures of Acinomadura verrucosospora before the onset of esperamicin synthesis inhibited the production of esperamicin A1 by the microorganism. This result indicates that esperamicin A1 is biosynthesized in part by the polyketide pathway. Addition of cerulenin to the cultures during the active production phase led to a net decrease in esperamicin A1 production. The 14C-acetate labeling pattern of esperamicin A1 in the cultures with or without addition of cerulenin at the active production phase also demonstrated the instability of esperamicin A1 in the fermentation. This suggests that esperamicin A1 is unstable and degradation occurs during the active production phase. Addition of the neutral resin Diaion HP-20 (1%) to the fermentation enhanced the production of esperamicin A1 by 53%.