1.-Lyxose was prepared from ribitol by a new method comprising a potent microbial oxidation reaction to convert ribitol to L-l'ibl.l|OSC, epimerization of the L-ribnlose to L-xylulose, and isomerization of the 1.-xylulose to produce L-lyxose. The complete transformation of ribitol to 1.-ribulosc was achieved using washed cells of Acetobacter aceti IFO 3281 at high substrate concentrations ranging from 5—20%. The 1.-ribulose produced was then used as the substrate [or the production of r-lyxose using immobilized L-rharnnose isomerase (r.-RI) of Pseudomonas sp. strain LL172 and immobilized 1)-tagatose 3-epimerase (n-TE) of recombinant Escherichia coli JM 105. At equilibrium, the yield of L-lyxose from 1.-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. 17221. 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.