Abstract Two β-mannanases (M-I and M-II) were purified from the culture filtrate of Enterococcus casseliflavus FL2121 by ammonium sulfate precipitation, column chromatography of DEAE-Toyopearl 650M and Phenyl-Toyopearl 650M and preparative polyacrylamide gel electrophoresis to homogenities. The molecular weights of M-I and M-II were estimated to be 142,000 and 137,000 by SDS-polyacrylamide gel electrophoresis and 127,000 and 113,000 by gel filtration, respectively. M-I and M-II exhibited an optimum pH at 6.0 and an optimum temperature at 50°C. The enzymes were activated slightly by CoCl 2 and MnCl 2, and inhibited strongly by AgNO 3, HgCl 2, EDTA, and N-bromosuccinimide. The K m values of M-I and M-II for konjac β-1,4-glucomannan were 0.14 and 0.30 (mg/ml), and the maximum velocities were 1,110 and 1,700 (U/mg protein), respectively. Both enzymes were endo-type β-mannanases hydrolyzing mannosides larger than β-1,4- d-mannotetraose.