The thermophilic anaerobe Thermoanaerobacterium saccharolyticum JW/SL-YS485 was investigated as a host for n-butanol production. A systematic approach was taken to demonstrate functionality of heterologous components of the clostridial n-butanol pathway via gene expression and enzymatic activity assays in this organism. Subsequently, integration of the entire pathway in the wild-type strain resulted in n-butanol production of 0.85g/L from 10g/L xylose, corresponding to 21% of the theoretical maximum yield. We were unable to integrate the n-butanol pathway in strainslacking the ability to produce acetate, despite the theoretical overall redox neutrality of n-butanol formation. However, integration of the n-butanol pathway in lactate deficient strains resulted in n-butanol production of 1.05g/L from 10g/L xylose, corresponding to 26% of the theoretical maximum.