Thermoanaerobacter ethanolicus is a xylose-utilizing thermophilic anaerobe that produces considerable amounts of ethanol. A protein in xylose-growing cells was solubilized from cell membranes by extraction with octyl-beta-glucoside. Internal peptide sequencing revealed that the protein was the product of a gene, xylF, encoding a putative D-xylose-binding protein. Metabolic labeling with 14C palmitic acid suggested that this is a lipoprotein that is anchored to the cell membrane via a cysteine residue. Binding was highly specific for xylose as evident by the lack of competition by sugars with structures similar to xylose. The apparent Kd of the protein for xylose was approximately 1.5 &mgr;M, and this value was very similar to the affinity constant determined for xylose transport by whole cells at low substrate concentrations. Uptake experiments with cells also suggested the presence of a separate low-affinity system. Binding activity varied less than 20% over a pH range of 4-8, and the level of activity was virtually unaffected when temperature was varied between 40 degreesC and 80 degreesC. This is the first biochemical characterization of a D-xylose-binding protein from a thermophilic organism.