D-Ribose, a five-carbon sugar, is used as a key intermediate for the production of various biomaterials, such as riboflavin and inosine monophosphate. A high D-ribose-producing Bacillus subtilis SPK1 strain was constructed by the chemical mutation of the transketolase-deficient strain, B. subtilis JY1. Batch fermentation of B. subtilis SPK1 with 20 g l(-1) xylose and 20 g l(-1) glucose resulted in 4.78 g l(-1) dry cell mass, 23.0 g l(-1) D-ribose concentration, and 0.72 g l(-1) h(-1) productivity, corresponding to a 1.5- to 1.7-fold increase when compared with values for the parental strain. A late-exponential phase was chosen as the best point for switching to a fed-batch process. Optimized fed-batch fermentation of B. subtilis SPK1, feeding a mixture of 200 g l(-1) xylose and 50 g l(-1) glucose after the late-exponential phase reduced the residual xylose and glucose concentrations to less than 7.0 g l(-1) and gave the best results of 46.6 g l(-1) D-ribose concentration and 0.88 g l(-1) h(-1) productivity which were 2.0- and 1.2-fold higher than the corresponding values in a simple batch fermentation.