Poly(gamma-glutamic acid) (PGA) produced by Bacillus subtilis F-2-01 on an industrial scale was ultimately depolymerized into a peptide fraction (F-I) and a gamma-glutamyl oligopeptide fraction (F-II) by the action of fungal PGA-hydrolase (gamma-L-glutamyl hydrolase). F-I consisted of both D- and L-glutamic acid (76:24), whereas L-glutamic acid alone was detected with F-II. Incomplete depolymerization of PGA depended on the presence of D-and L-glutamic acid residues in F-I but not on end product inhibition or denaturation of the enzyme. Analyses by ultracentrifugation supported the loss of intermolecular interaction between gamma-glutamyl peptides that may interfere with the enzymatic hydrolysis of F-I by accelerating aggregate formation. F-I is proposed to exist as an optically heterogeneous peptide unit in PGA, in which D-and L-glutamic acid isomers are copolymerized into a single chain.