Polymers of basic amino acids function as polycationic compounds under physiological conditions and exhibit intriguing biological properties, such as antimicrobial and antiviral activities, immunopotentiating ability, and DNA-binding activity. Poly(epsilon-l-lysine) (epsilon-PL) produced by some strains of Streptomyces spp. is a cationic homopolymer of l-lysine linking between epsilon-amino and alpha-carboxylic acid functional groups and has been used as a food preservative based on its biocompatibility and biodegradability. An epsilon-PL-producing strain of Streptomyces sp. USE-33 was found to secrete a novel polycationic substance into its culture broth along with epsilon-PL. High-performance liquid chromatography analyses and one- and two-dimensional H-1 and C-13 nuclear magnetic resonance (NMR) experiments, accompanied by NMR titration studies, revealed that the secreted substance was poly[beta-(l-diaminopropionyl-l-diaminopropionic acid)], PAP, characterized by an isopeptide backbone linking between the beta-amino and alpha-carboxylic acid groups of l-alpha,beta-diaminopropionic acid (l-Dpr) with pendent l-Dpr residues. PAP had a molecular weight of 500 to 1400, and copolymers composed of the two amino acids l-Dpr and l-lysine were not detected in the producer strain USE-33. The strain coproduced high levels of the two poly(amino acid)s in the presence of glycerol, citrate, and ammonium sulfate at pH 4.0 in a two-stage cultivation procedure. PAP exhibited strong inhibitory activities against several yeasts and weaker activities against bacteria than epsilon-PL. PAP may share a number of biological functions with epsilon-PL, and the use of PAP along with epsilon-PL has potential as a specific and advanced material for technical applications in various fields.