L-Lyxose was prepared from ribitol by a. new method comprising a potent microbial oxidation reaction to convert ribitol to L-ribulose, epimerization of the L-ribulose to L-xylulose, and isomerization of the L-xylulose to produce L-lyxose. The complete transformation of ribitol to L-ribulose was achieved using washed cells of Acetobacter aceti IFO 3281 at high substrate concentrations ranging from 5-20%. The L-ribulose produced was then used as the substrate for the production of L-lyxose using immobilized L-rhamnose isomerase (L-RI) of Pseudomonas sp. strain LL172 and immobilized D-tagatose 3-epimerase (D-TE) of recombinant Escherichia coli JM 105. At equilibrium, the yield of L-lyxose from L-ribulose was determined to be about 60%, and the product could be isolated easily from the reaction mixture after degradation of ketoses using Pseudomonas sp. 172a. Following various product purification steps, about 5.0 g L-lyxose crystals were recovered from 10.0 g ribitol in a flask reaction. The crystallized product was finally identified by HPLC, IR spectrum, NMR, and optical rotation measurements.