The effects of C-14-labelled precursor feeding on the production of cyclic hexadepsipeptides were investigated by the mycelium of F. oxysporum KFCC11363P producing beauvericin along with enniatins H, I, and MK1688. Most C-14-phenylalanine and C-14-valine were incorporated easily into the biosynthetic pathway of C-14-labelled beauvericin in vivo by the mycelium. However, only a small amount of C-14-labelled enniatins could be detected by feeding of C-14-valine. When L-valine was fed as a precursor to the mycelium at large scale, the level of beauvericin increased to maximum followed by enniatins H and I. Feeding of L-valine did not affect the production of enniatin MK1688. These results suggest that L-valine feeding led to the production of D-hydroxyisovaleic acid in the mycelium and specifically enhanced the production of cyclic hexadepsipeptides containing D-hydroxyisovaleic acid, such as beauvericin and enniatins H and I. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.