gamma-Glutamyltranspeptidase (GGT) from Bacillus subtilis is an extracellular enzyme that exhibits glutaminase activity and is thus suitable for the fermentation of foods. As GGT of B. subtilis is synthesized only during the mid-stationary phase, which is inconvenient for industrial use, a strain overexpressing GGT for a sufficiently long period was generated to obtain large quantities of GGT. A plasmid vector, pHY300PLK, containing the ggt gene cloned from chromosomal DNA was introduced into a spo0A abrB double mutant strain, in which the level of GGT activity is high after the mid-stationary phase. The level of GGT activity in this strain increased steadily after the exponential phase, becoming 15-fold higher than that in the parental strain. The recombinant GGT was purified by 252-fold with a yield of 30.4%. The enzyme is a heterodimer consisting of one large subunit (45 kDa) and one small subunit (21 kDa). The enzyme is highly salt-tolerant and converts glutamine to glutamic acid effectively even in the presence of 18% NaCl. This is the first report of salt-tolerant GGT.