ε-Poly- l -lysine (ε-PL) is produced by Streptomyces albulus NBRC14147 as a secondary metabolite and can be detected only when the fermentation broth has an acidic pH during the stationary growth phase. Since strain NBRC14147 produces ε-PL-degrading enzymes, the original chain length of the ε-PL polymer product synthesized by ε-PL synthetase (Pls) is unclear. Here, we report on the identification of the gene encoding the ε-PL-degrading enzyme (PldII), which plays a central role in ε-PL degradation in this strain. A knockout mutant of the pldII gene was found to produce an ε-PL of unchanged polymer chain length, demonstrating that the length is not determined by ε-PL-degrading enzymes but rather by Pls itself and that the 25 to 35 l -lysine residues of ε-PL represent the original chain length of the polymer product synthesized by Pls in vivo . Transcriptional analysis of pls and a kinetic study of Pls further suggested that the Pls catalytic function is regulated by intracellular ATP, high levels of which are required for full enzymatic activity. Furthermore, it was found that acidic pH conditions during ε-PL fermentation, rather than the inhibition of the ε-PL-degrading enzyme, are necessary for the accumulation of intracellular ATP.