The spent liquor from acidic sulphite pulping of Eucalyptus globulus (HSSL) is the side product from sulphite pulp production and besides sulphonated lignin contains sugars from degraded hemicelluloses, plainly pentoses. Pichia stipitis fermentation of these sugars for bioethanol production was the primary goal of this work. The increasing of HSSL proportion in fermentation media affected negatively the ethanol yield. Thus with 20% of HSSL (v/v) attained maximum ethanol yield was 0.15 g of ethanol by g of sugar consumed (g(e) g(s)(-1)) and with 60% (v/v) only 0.08 g(e) g(s)(-1). Biological removal of acetic acid from HSSL improved fermentation though the complete removal of acetic acid and polyphenolics (including sulphonated species) by treatment with ion-exchange resins was required for highly successful bioethanol production. Accordingly, the fermentative metabolic pathway of P. stipitis has been promoted allowing fair ethanol productivity and yield (Yp/s=0.49g(e)g(s)(-1)) at relatively low maximum of cell growth rate (mu(max)=0.21 h(-1)). (C) 2009 Elsevier Ltd. All rights reserved.