A polyglutamyl gamma-hydrolase enzyme has been identified which catalyses the hydrolytic breakdown of poly-gamma-D-glutamic acid (PGA) from Bacillus licheniformis 9945a. The enzyme was found to be physically associated with the polymer and was activated by Zn2+ or Ca2+ salts. The enzyme can be solubilized from the polymer by treatment with 0.5% SDS and 1 mM ZnCl2 and can then be renatured onto exogenous PGA upon dilution below the detergent critical micellar concentration. The enzyme was partially purified by affinity chromatography, using immobilized PGA. Peptide thioesters containing one and two gamma-glutamyl units were synthesized as potential chromogenic substrates but showed no activity with the solubilized enzyme. Examination of 14C-labeled reaction products indicated that the enzyme is an endo-type hydrolase.