Abstract: In the cotyledons of germinating Lentil (Lens esculenta L.) at 15 days the abundant amylolytic activity was found to be β-amylase (E.C. 3.2.1.2, α-1-4-glucan maltohydrolase). β-amylase from germinating Lentil cotyledons was purified to homogeneity for study of enzyme characteristics. The purification steps included ammonium sulphate precipitation followed by DEAE-cellulose chromatography and hydroxylapatite chromatography. By successive steps of (NH4)2 SO4 fractionation, DEAE-cellulose chromatography and HA filtration, the purity of the enzyme from lentil increased 108.5 fold with an overall yield of 32.2% with specific activity of 575 U mg-1. The end product of -1, 4-glucan degradation was maltose. The β-amylase was most active at pH 6.1 (soluble starch substrate) and 40°C. Km for Lentil cotyledons β-amylase for soluble potato starch was 1.67 milligrams per milliter (mg ml-1). The molecular weight of the enzyme was estimated to be 58 kilodaltons (kDa) by both gel filtration and sodium dodecyl sulphate gel electrophoresis. Heavy-metal ions Cu2+, Hg2+ and Ag+ at 1.0 mM concentration and p-hydroxymercuribenzoic acid and N-bromosuccinimide at 0.4 mM concentration inhibited the enzyme activity. Lentil β-amylase is competitively inhibited by its end product, maltose, with a Ki of 8.4 mM. Less branched or no-branched (amyloses) were better substrates for Lentil cotyledons β-amylase than moderately branched glucans ( amylopectin) or highly branched (glycogens) glucans.