In Escherichia coli, classified as a mixed-acid producer in fermentation, D-lactate is one of the final metabolites from pyruvate. In order to achieve a high efficiency of bacterial hydrogen production from glucose, we have constructed an E. coli strain deficient in fermentative lactate dehydrogenase (LDH-A) by P1 transduction. The mutant, designated as MC13-4, entirely lost LDH-A activity while retaining whole formate hydrogenlyase activity. This mutation resulted in an increase in hydrogen production based on glucose consumed. The effect of uptake hydrogenases on the hydrogen production was also discussed.