The biosynthesis of poly(ɛ-l-lysine) (ɛ-PL) in the two newly isolated strains of Streptomyces lydicus USE-11 (USE-11) and Streptomyces sp. USE-51 (USE-51) was studied by a newly developed two-stage culture method of cell growth at pH 6.8 and ɛ-PL production at pH 4.5. USE-11 synthesized ɛ-PL consisting of about 28 residues at a high production level, whereas USE-51 did the polymer with 15 ones at a low level. The secreted ɛ-PLs in culture media were digested in a neutral pH range with a peptide hydrolase(s) produced by the ɛ-PL producers. The optimum production levels were presumed to be dependent upon the inherent ɛ-PL synthesis machinery of each producer. The production in USE-51 was sharply dependent upon cell density as was often observed in the production of antibiotics, whereas that in USE-11 was scarcely affected by the density. The % MathType!Translator!2!1!AMS LaTeX.tdl!TeX -- AMS-LaTeX!% MathType!MTEF!2!1!+-% feaaeaart1ev0aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbbjxAHX% garmWu51MyVXgatuuDJXwAK1uy0HwmaeHbfv3ySLgzG0uy0Hgip5wz% aebbnrfifHhDYfgasaacH8qrps0lbbf9q8WrFfeuY-Hhbbf9v8qqaq% Fr0xc9pk0xbba9q8WqFfea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qq% Q8frFve9Fve9Ff0dmeaabaqaciGacaGaaeqabaWaaeWaeaaakeaaca% qGtbGaae4tamaaDaaaleaacaqG0aaabaGaaeOmaiaab2caaaaaaa!3B81! $${\text{SO}}^{{{\text{2 - }}}}_{{\text{4}}}$$ was found to be essential for the ɛ-PL production in both strains. This might suggest the involvement of a thiol group in the polymerization reactions including the activation of l-lysine. This study indicates that USE-11 is a most suitable strain for the exploration of the ɛ-PL biosynthesis at the molecular level as well as for the technical applications.