Bifidobacterium pseudolongum NCFB 2244 grew on starch as sole source of carbon and energy, but cell yields and specific growth rates were considerably lower than on glucose (mu = 0.19 +/- 0.04 and 0.38 +/-0.09 respectively). Amylase activity was not detected in cultures of this bacterium, but cell-associated alpha-glucosidase was constitutively produced. Analysis of alpha-glucosidase activity from cell extracts by preparative isoelectric focusing gave two peaks of activity with apparent isoelectric points of 3.9 (Enzyme I) and 4.2 (Enzyme II), corresponding to threefold and fourfold purification factors respectively. No alpha-glucosidase activity was detected with Enzyme I after gel-filtration chromatography on Sephadex G150. However, activity was recovered in samples containing Enzyme II, indicating the protein had a molecular mass of approximately 126 kDa. This was subsequently confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). These results show that the restricted ability of B. pseudolongum to utilize starch as a carbohydrate source is owing to synthesis of at least one, and possibly two, alpha-glucosidase(s).