The alpha-glucosidase enzyme was isolated from vegetative cells and spores of Bacillus stearothermophilus, ATCC 7953. Spore-associated enzyme had a molecular weight of approximately 92 700, at temperature optimum of 60 °C, and a pH optimum of 7·0-7·5. The enzyme in crude aqueous spore extract was stable for 30 min up to a temperature of 65 °C, above which the enzyme was rapidly denatured. The optimal pH for stability of the enzyme was approximately 7·2. The alpha-glucosidase in crude vegetative cell extract had similar characteristics to the spore-associated enzyme but its molecular weight was 86 700. The vegetative cell and spore-associated enzymes were cross-reactive. The enzymes are postulated to derive from a single gene product, which undergoes modification to produce the spore-associated form. The location of alpha-glucosidase in the spore coats (outside the spore protoplast) is consistent with the location of most enzymes involved in activation, germination and outgrowth.