In this study, we attempted to purify and characterize glutaminase (EC. 3.5.1.2) from Lactobacillus reuteri KCTC3594. The glutaminase was purified approximately 21-fold from the cell-free extract of L. reuteri KCTC3594 by protamine sulfate treatment and chromatography methods including anion exchange and gel filtration. The sizes of two major bands of the enzyme were presumed to be 70 and 50 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The glutaminase activity of L. reuteri KCTC3594 was assayed in various ranges of pH, temperature, and salt concentrations. The enzyme activity was optimal at 40 degrees C and pH of 7.5. It was shown that the glutaminase was salt-tolerant because the enzyme activity was maintained 50% at 15% (w/v) salt concentrations. On the other hand, the enzyme was strongly inhibited up to 80% by 6-diazo-5-oxo-L-norleucine (10 mM) and iodoacetate (50 mM) indicating that the purified enzyme represents typical characteristics of glutaminase.