alpha-Glucosidase (EC 3.2.1.20) was purified to homogeneity from logarithmically growing cells of Saccharomyces carlsbergensis. The purification involved the following steps: (a) ammonium sulfate fractionation; (b) Sephadex G-100 chromatography; (c) DEAE-cellulose chromatography; and (d) hydroxylapatite chromatography. This procedure gave a preparation judged to be greater than 98% pure by Na-DodSO4-polyacrylamide gel electrophoresis. The enzyme was shown to be a monomer of 63 000 daltons by gel filtration on Sephacryl S-200 under native conditions and by polyacrylamide gel electrophoresis under denaturing conditions. The Km values of the enzyme for the substrates maltose and p-nitrophenyl alpha-D-glucoside were found to be 1.66 X 10(-2) and 3.1 X 10(-4) M, respectively. The corresponding Vmax value for maltose was 44.8 X 10(-6) mol min(-1) mg(-1) and that for p-nitrophenyl alpha-D-glucoside was 134 X 10(-6) mol min-1 mg-1. The pH optimum for the purified enzyme was found to be between pH 6.7 and 6.8. The enzyme has an absolute anomeric specificity for alpha-glycosidic linkages and appears to recognize a glucosyl residue in alpha linkage on the nonreducing end of its substrate. For the strain used in this study, which carries the MAL 6 locus, only a single form of the enzyme was detected.