Glutamate dehydrogenase from Giardia intestinalis was purified 680-fold to electrophoretic homogeneity with a 42% recovery through a two-step procedure. The most effective step in the purification was the use of CM-Trisacryl that eliminated nearly 99% of the total proteins with 100% recovery. Matrix-assisted laser desorption ionization time-of-flight mass spectrometer was used to analyze the giardial glutamate dehydrogenase after deposition of the purified enzyme on a crystalline layer of 3,5-dimethoxy-4-hydroxy-trans-cinnamic acid. Use of this sample preparation technique allowed the first successful determination of the molecular mass of the enzyme (50,120 +/- 75). Since the molecular weight of the native enzyme was determined to be 270,000 by gel filtration, the enzyme appears to be a hexamer. The enzyme was specific for NADP(H) and functioned more favorably in the direction of glutamate formation than catabolism. The pH optimum was 7.5 for reductive amination of 2-oxoglutarate and 9.3 for oxidative deamination of glutamate. The apparent K(m) values were 0.28 mM for 2-oxoglutarate and 17 microM for NADPH. An unusual biphasic saturation curve characterized the effect of ammonium ion on the activity with a plateau between 40 and 55 mM.