The ethanologenic bacteria Escherichia coli strains KO11 and LYO1, and Klebsiella oxytoca strain P2, were investigated for their ability to metabolize furfural. Using high performance liquid chromatography and 13C-nuclear magnetic resonance spectroscopy, furfural was found to be completely biotransformed into furfuryl alcohol by each of the three strains with tryptone and yeast extract as sole carbon sources. This reduction appears to be constitutive with NAD(P)H acting as electron donor. Glucose was shown to be an effective source of reducing power. Succinate inhibited furfural reduction, indicating that flavins are unlikely participants in this process. Furfural at concentrations >10 mM decreased the rate of ethanol formation but did not affect the final yield. Insight into the biochemical nature of this furfural reduction process may help efforts to mitigate furfural toxicity during ethanol production by ethanologenic bacteria.