The genome of Clostridium acetobutylicum 824 contains two genes encoding NAD, Mn(2+) ion, and dithiothreitol-dependent phospho-alpha-glucosidases that can be assigned to Family 4 of the glycosylhydrolase superfamily. The two genes, designated malh (maltose 6-phosphate hydrolase) and pagl (phospho-alpha-glucosidase) respectively, reside in separate operons that also encode proteins of the phosphoenolpyruvate-dependent:sugar phosphotransferase system (PEP-PTS). C. acetobutylicum grows on a variety of alpha-linked glucosides, including maltose, methyl-alpha-D-glucoside and the five isomers of sucrose. In the presence of the requisite cofactors, extracts of these cells readily hydrolyzed the chromogenic substrate p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNPalphaGlc6P), but whether hydrolysis reflected expression of enzymes encoded by the malH or pagL genes was not discernable by spectro- photometric analysis or polyacryl- amide gel electrophoresis. Resolution of this question required the cloning of the malH and pagL genes, and subsequent high-expression, purification, and characterization of maltose 6-phosphate hydrolase (MalH) and phospho-alpha-glucosidase (PagL), respectively. Both MalH and PagL are tetramers comprised of similar?sized (~50 kDA) subunits, and they exhibit 50% residue identity. The two proteins cross-react with polyclonal rabbit antibody against phospho-alpha-glucosidase from Fusobacterium mortiferum. Although purified MalH and PagL cleaved pNPaGlc6P with comparable efficiency, only MalH catalyzed the hydrolysis of disaccharide 6-phosphates formed via the PEP-PTS. Analysis of the proteome of C. acetobutylicum by electrospray ionization/mass spectrometry revealed induction of MalH during growth on all alpha-glucosides tested, whereas PagL was detected only in extracts prepared from organisms grown previously on maltose and methyl-alpha-D-glucoside. Our studies provide evidence: (i) that proteins encoded by the mal operon facilitate the transport and dissimilation of maltose and related O-alpha-linked glucosides, by C. acetobutylicum 824; (ii) that a conserved cysteine residue is required for phospho-alpha-glucosidase activity, and (iii) that dithiothreitol may contribute to enzyme stability by preventing the formation of inter- and intra -subunit disulfide bonds.