Antimicrobial epsilon-poly-l-lysine (ePL) is secreted by Streptomycetaceae bacteria, and the mechanism of ePL biosynthesis remains to be elucidated. We previously reported that an unknown ePL derivative accumulates in the culture medium of ePL-producing bacteria when glycerol is added to the culture medium (Nishikawa and Ogawa, Appl. Environ. Microbiol. 68:3575-3581, 2002). In this study, by using matrix-assisted laser desorption ionization—time of flight mass spectrometry and nuclear magnetic resonance, we identified the unknown derivative as the ester formed between the hydroxyl group of a glycerol molecule and the terminal carboxyl group of an ePL molecule. When a short-chain aliphatic polyol, such as ethylene glycol, propanediol, or butanediol, was added instead of glycerol, a corresponding ePL-polyol monoester accumulated in the culture medium of ePL-producing bacteria. ePL esterification was accompanied by ePL synthesis in intact cells and a cell-free system, but no esterification of exogenous ePL was observed. ePL-polyol esters were formed during lysine polymerization. The number of lysine residues of ePL-polyol esters decreased with increasing polyol concentration. Taken together, these results indicate that ePL synthesis is inhibited by polyols via esterification and that ePL elongation occurs via the incorporation of lysine monomers into the carboxyl terminus of ePL.