A genetic locus from Staphylococcus xylosus involved in maltose-maltotriose utilization has been characterized. The chromosomal region was identified by screening a genomic library of S. xylosus in Escherichia coli for sucrose hydrolase activity. Nucleotide sequence analysis yielded two open reading frames (malR and malA) encoding proteins of 37.7 and 62.5 kDa, respectively. MalR was found to be homologous to the LacI-GalR family of transcriptional regulators, and MalA showed high similarity to yeast alpha-1,4-glucosidases and bacterial alpha-1,6-glucosidases. Inactivation of malA in the genome of S. xylosus led to a maltose-maltotriose-negative phenotype. In cell extracts of the mutant, virtually no glucose release from maltose and short maltodextrins was detectable. Inactivation of malA in a sucrose-6-phosphate hydrolase-deficient S. xylosus strain resulted in the complete loss of the residual sucrose hydrolase activity. The MalA enzyme has a clear preference for maltose but is also able to release glucose from short maltosaccharides. It cannot cleave isomaltose. Therefore, malA encodes an alpha-1,4-glucosidase or maltase, which also liberates glucose from sucrose. Subcloning experiments indicated that malA does not possess its own promoter and is cotranscribed with malR. Its expression could not be stimulated when maltose was added to the growth medium. Chromosomal inactivation of malR led to reduced maltose utilization, although alpha-glucosidase activity in the malR mutant was slightly higher than in the wild type. In the mutant strain, maltose uptake was reduced and inducibility of the transport activity was partially lost. It seems that MalR participates in the regulation of the gene(s) for maltose transport and is needed for their full expression. Thus, the malRA genes constitute an essential genetic locus for maltosaccharide utilization in S. xylosus