Xylitol was used as a raw material for production Of L-xylose and L-lyxose using Alcaligenes 701B strain and immobilized L-rhamnose isomerase enzyme. Alcaligenes 701B converted Xylitol to L-xylulose with a yield of 34% in the bioreactor. L-xylulose was converted to L-xylose and L-lyxose using immobilized L-rhamnose isomerase enzyme. The final equilibrium between L-xylulose, L-xylose and L-lyxose was 53:26:21. The enzyme assays indicated that Alcaligenes 701B strain has an NAD-dependent xylitol dehydrogenase enzyme responsible for L-xylulose production. Furthermore, NAD(P)H-dependent L-xylulose reductase enzyme was active during conversion of xylitol to L-Xylulose. The highest L-xylulose production rate corresponded with the highest growth rate. The Alcaligenes 701B strain used D-Xylose for biomass growth, but xylitol was used only for L-xylulose production during conversion phase. (c) 2005 Elsevier Inc. All rights reserved.