beta-Phosphoglucomutase (beta-PGM, EC 5.4.2.6) was isolated to homogeneity from a cell-free extract of Lactococcus lactis subsp. cremoris IFO 3427 by chromatographies with QAE-Sephadex A-50, phenyl-Sepharose CL-4B, hydroxylapatite, and Bio-Gel A-1.5m. The enzyme was purified about 260-fold with a yield of 7.2% and a specific activity of 113 units/mg protein. The molecular weight was estimated to be 34,000 and 25,000 by HPLC gel filtration on TSI(gel G3000SW(XL) and SDS-PAGE, respectively. The enzyme showed optimum activity around pH 7.0 and its optimum temperature was about 40 degrees C. The enzyme was stable over a pH range from 5.0 to 9.5 and retained its activity up to 45 degrees C. It was activated by four divalent cations (Co2+ >Mn2+ > Mg2+ >Ni2+ at 1.0 mM concentration). The K-m value was 0.23 mM for beta-D-glucose 1-phosphate. The enzyme activity was strongly inhibited by other divalent cations (Cu2+, Cd2+, Zn2+, and Hg2+). ADP and ATP also greatly inhibited the enzyme activity, whereas AMP hardly did. alpha-D-Glucose l-phosphate and D-glucose 6-phosphate were not potent inhibitors of the enzyme. A comparison of its characteristics with the properties of other known beta-PGMs indicated that the beta-PGM from Lactococcus lactis subsp. cremoris IFO 3427 is a new type of enzyme.