Glutathione (gamma-glutamyl-L-cysteinylglycine, GSH) is a pharmaceutical compound often used in food additives and the cosmetics industry. GSH can be produced biologically from L-glutamic acid, L-cysteine, and glycine through an enzymatic process traditionally involving two sequential adenosine triphosphate (ATP)-dependent reactions catalyzed by gamma-glutamylcysteine synthetase (gamma-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). Here, we report the enzymatic production of GSH by recombinant cell-free bifunctional gamma-glutamylcysteine synthetase/glutathione synthetase (gamma-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation. GSH production by an acetate kinase-integrated Escherichia coli Rosetta(DE3) mutant expressing Streptococcus thermophilus GshF reached 18.3 +/- A 0.1 g l(-1) (59.5 +/- A 0.3 mM) within 3 h, with a molar yield of 0.75 +/- A 0.00 mol mol(-1) added cysteine and a productivity of 6.1 +/- A 0.0 g l(-1) h(-1). This is the highest GSH titer reported to date. This newly developed biocatalytic process offers a promising approach for meeting the industrial requirements for GSH production.