The production of ethanol by Pichia stipitis CBS 5773 in semi-synthetic xylose-containing media and in sugar cane bagasse hemicellulosic hydrolysate was investigated. The effect of xylose concentration upon fermentation was determined for P. stipitis and three other yeasts. An effective method of hydrolysate treatment employing neutralization with NaOH and activated charcoal was established. Fermentation of hydrolysate thus treated showed an extended lag phase and a 20% lower maximum volumetric productivity (i.e., 0·82 g l−1 h−1) relative to fermentation of semi-synthetic media; however, yield (0·35 g g−1) and final ethanol concentration (24 g l−1) did not differ. Recycling of the yeast four times in treated hydrolysate-containing medium did not improve efficiency.