Abstract Bacillus stearothermophilus produces low levels of ethanol following fermentation of glucose at temperatures in excess of 60°C. A major by-product is l-lactic acid. To improve ethanol yields mutants of this organism were isolated which lacked NAD-linked l-lactate dehydrogenase, the enzyme responsible for lactic acid production. This was achieved by selection for resistance to a suicide substrate, fluoropyruvate. Such mutants no longer produced lactic acid and yields of ethanol from glucose were increased by at least a factor of two.