In the process of producing ethanol from lignocellulosic materials such as wheat straw, compounds that can act inhibitory to enzymatic hydrolysis and to cellular growth may be generated during the pretreatment. Ethanol production was evaluated on pretreated wheat straw hydrolysate using four different recombinant Saccharomyces cerevisiae strains, CPB.CR4, CPB.CB4, F12, and FLX. The fermentation performance of the four S. cerevisiae strains was tested in hydrolysate of wheat straw that has been pretreated at high dry matter content (220 g/L dry matter). The results clearly showed that F12 was the most robust strain, whereas the other three strains were strongly inhibited when the fraction of hydrolysate in the fermentation medium was higher than 60% (v/v). Furthermore, the impact of different lignin derivatives commonly found in the hydrolysate of pretreated wheat straw, was tested on two different enzyme mixtures, a mixture of Celluclast 1.5 L FG and Novozym 188 (3:1) and one crude enzyme preparation produced from Penicillium brasilianum IBT 20888. From all the potential inhibiting compounds that were tested, formic acid had the most severe influence on the hydrolysis rate resulting in a complete inactivation of the two enzyme mixtures. Biotechnol. Bioeng. 2007;96: 250–258. © 2006 Wiley Periodicals, Inc.