The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by amyX, in the carbohydrate metabolism of Bacillus subtilis 168 were investigated using yvdF, amyX, and yvdF amyX mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and beta-cyclodextrin (beta-CD) taken up by wild-type B. subtilis cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the yvdF mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and beta-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched beta-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a glg mutant with a wild-type background, yvdF glg and amyX glg mutants, and a glg mutant with a double mutant (DM) background. The amyX glg and glg DM strains accumulated significantly larger amounts of glycogen than the glg mutant, while the yvdF glg strain accumulated an intermediate amount. Glycogen samples from the amyX glg and glg DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the glg mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the alpha-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.