Stillage is the main residue from the starch-to-ethanol fermentation process. Carbohydrates (hemicellulose and cellulose) comprise approximately 50% (w/w) of the total components of stillage. Conversion of the hemicellulose and cellulose to fermentable sugars and then to ethanol has the potential to significantly increase the efficiency of the process. The hydrolysis of stillage to fermentable sugars was optimised using 2% (v/v) H2SO4 at View the MathML source for 5.5 h and produced 18 g/L xylose, 11.5 g/L arabinose and 6.5 g/L glucose from 120 g/L stillage. Further hydrolysis using enzymes increased the release of glucose by 61%. Furfural, acetate and lactate were the main inhibitors present in the acid hydrolysate of stillage. The lignin-derived inhibitors hydroxymethylfuraldehyde, hydroxybenzaldehyde, vanillin and syringaldehyde were not detected. Neutralisation of the hydrolysate with lime to pH 5 decreased the concentration of furfural by 50%. Fermentation of hydrolysate supplemented with glucose 10 g/L, by recombinant Zymomonas mobilis ZM4(pZB5), produced 11 g/L of ethanol after 70 h, with residual xylose 12 g/L. Supplementation of the hydrolysate with 5 g/L yeast extract and 40 g/L glucose produced 28 g/L ethanol with 2.6 g/L residual xylose after 18 h. Arabinose was not utilised by this particular recombinant strain. From the results, Z. mobilis ZM4(pZB5) may be a suitable candidate for the fermentation of both glucose and xylose in stillage acid hydrolysates.