The effects of glycerol and the oxygen transfer rate on the xylitol production rate by a xylitol dehydrogenase gene (XYL2)-disrupted mutant of Candida tropicalis were investigated. The mutant produced xylitol near the almost yield of 100% from D-xylose using glycerol as a co-substrate for cell growth and NADPH regeneration: 50 g D-xylose l(-1) was completely converted into xylitol when at least 20 g glycerol l(-1) was used as a co-substrate. The xylitol production rate increased with the O-2 transfer rate until saturation and it was not necessary to control the dissolved O-2 tension precisely. Under the optimum conditions, the volumetric productivity and xylitol yield were 3.2 g l(-1) h(-1) and 97% (w/w), respectively.