Methods of achieving high-yield fermentation of D-xylose to ethanol in shake flasks were investigated using the microaerophilic yeast Pichia stipitis NRRL Y-7124. Ethanol yields based on consumed xylose approached the theoretical maximum of 0.51 g/g at high culture volumes. Although volumetric productivities decreased from 0.46 g/L-h at 100 mL to 0.15 g/L-h at 225 mL, ethanol yields on consumed xylose of 0.50 g/g were achieved in 250-mt DeLong culture flasks at culture volumes of 200 and 225 mt.