Conditions have been optimized for fermentation of pretreated hardwood spent sulfite liquor (HSSL) using an adapted strain of Pichia stipitis. The pretreatments, consisting of boiling and overliming with Ca(OH)2 of HSSL, to partially remove inhibitors, and adaptation of the yeast strain to HSSL, were both critical for a successful fermentation. Ethanol concentration was increased from 6.7 to 20.2 g l(-1) using adapted P. stipitis (A) and pretreated HSSL. The maximum ethanol yield (Yp/s) and productivity (Qp) were 0.41 g g(-1) and 0.44 g l(-1) h(-1), respectively, at an oxygen transfer rate of 2.0 mmol O2 l(-1) h(-1). The optimized results with this strain were compared to those of other xylose-fermenting yeasts and Saccharomyces cerevisiae (SSL-acclimatized) currently used at an industrial plant for the fermentation of spent sulfite liquor.