Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment D-ribitol (also called D-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates D-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in D-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented D-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a D-ribitol-5-phosphate (D-ribitol-5-P) 2-dehydrogenase, a D-ribulose-5-P 3-epimerase, a D-ribose-5-P isomerase, and a D-xylulose-5-P phosphoketolase. In the first catabolic step, the protein D-ribitol-5-P 2-dehydrogenase uses NAD(+) to oxidize D-ribitol-5-P formed during PTS-catalyzed transport to D-ribulose-5-P, which, in turn, is converted to D-xylulose-5-P by the enzyme D-ribulose-5-P 3-epimerase. Finally, the resulting D-xylulose-5-P is split by D-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate D-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as D-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation.