For the first time, we succeeded in synthesizing in vitro poly-gamma-glutamate (PGA) with high molecular masses (>1,000 kDa) by the use of enzyme-associated cell membranes from Bacillus subtilis subsp. chungkookjang. The activity for PGA synthesis, however, was readily lost in the presence of critical concentrations of detergents tested in micelles. The optimum pH for the reaction was found to be approximately 7.0. We examined the effects of some divalent cations on PGA synthesis and found that Mg(2+) was essential in catalysis and that Zn(2+) additionally boosted the activity. In contrast, Fe(2+) and Ca(2+) acted as inhibitors. Mn(2+) did not apparently influence the in vitro formation of PGA. DL-Glutamate (D isomer content, 60 to 80%) apparently served as the best substrate; d-Glutamate was preferable to the L isomer as a substrate. When D- and L-glutamate were used for the reaction, the elongated chains of PGAs were composed of the D- and L-isomers, respectively. Our results suggest that the stereochemical properties of enzymatically synthesized PGAs substantially depend on the stereochemistry (DL ratio) of glutamate as the substrate. Furthermore, genetic analysis indicated that all the pgsB, -C, and -A gene products, which are responsible for PGA production by B. subtilis cells, were also indispensable for enzymatic PGA synthesis.