A 7-kb DNA fragment of Lactobacillus sakei, containing the rbsD, rbsK and rbsR genes was sequenced. The genes responsible for ribose utilization are organized differently from what was previously described for model organisms such as Escherichia coli and Bacillus subtilis. No gene encoding RbsA, RbsB and RbsC, the subunits of the ribose ABC-transporter, were present in the rbs gene cluster. Instead, we found an open reading frame coding for RbsU, a protein similar to GltA, the glucose transporter of Staphylococcus xylosus. The disruption of rbsK, encoding the ribokinase, impaired growth on ribose. The disruption of rbsR, encoding the repressor, had no effect on the ability to grow on ribose, but led to overexpression of a large transcript corresponding to rbsU, rbsD and rbsK, suggesting that RbsU might be involved in ribose utilization. Ribose uptake and phosphorylation assays on the wild type strain and various mutants showed that, in ptsI mutants, both ribose uptake and phosphorylation are increased. These increased activities can explain the faster growth rate on ribose that was observed in ptsI mutants. The phosphotransferase system is thus involved in the negative regulation of ribose utilization. This regulation might not act at the transcriptional level since the overexpression of the rbs genes in the rbsR mutant did not lead to the same phenotype. A gene sharing high similarity scores with ackA genes, encoding the acetate kinase, was found upstream from the rbs gene cluster. The unusual location of this gene is maybe not fortuitous since acetate kinase is involved in ribose catabolism.