Thermophilic bacteria have gained increased attention as candidates for bioethanol production from lignocellulosic biomass. This study investigated ethanol production by Thermoanaerobacter strain J1 from hydrolysates made from lignocellulosic biomass in batch cultures. The effect of increased initial glucose concentration and the partial pressure of hydrogen on end product formation were examined. The strain showed a broad substrate spectrum, and high ethanol yields were observed on glucose (1.70 mol/mol) and xylose (1.25 mol/mol). Ethanol yields were, however, dramatically lowered by adding thiosulfate or by cocultivating strain J1 with a hydrogenotrophic methanogen with acetate becoming the major end product. Ethanol production from 4.5 g/L of lignocellulosic biomass hydrolysates (grass, hemp stem, wheat straw, newspaper, and cellulose) pretreated with acid or alkali and the enzymes Celluclast and Novozymes 188 was investigated. The highest ethanol yields were obtained on cellulose (7.5 mM·g(-1)) but the lowest on straw (0.8 mM·g(-1)). Chemical pretreatment increased ethanol yields substantially from lignocellulosic biomass but not from cellulose. The largest increase was on straw hydrolysates where ethanol production increased from 0.8 mM·g(-1) to 3.3 mM·g(-1) using alkali-pretreated biomass. The highest ethanol yields on lignocellulosic hydrolysates were observed with hemp hydrolysates pretreated with acid, 4.2 mM·g(-1).