Furfural and 5-hydroxymethylfurfural (HMF) are major inhibitory compounds generated from biomass pretreatment using dilute acid hydrolysis.  Remediation of inhibitors adds cost and generates extra waste products.  Few yeast strains tolerant to inhibitors are available and the need for tolerant strains is well recognized.  Understanding mechanisms of the tolerance and the in situ detoxification of the inhibitors is necessary for such tolerant strain development.  Furfural conversion to 2-furanmethanol (FM; furfural alcohol) is well studied and documented, and HMF metabolic conversion product was recently isolated and identified as 2,5-furan-dimethanol (FDM; 2,5-bis-hydroxymethylfuran).  Such identification allowed investigations on metabolic profiling and mechanisms of the inhibitor detoxification.  We investigated metabolic profiling of ethanologenic yeast in the presence of furfural and HMF and synthesized the HMF conversion product, FDM, which is not commercially available.  NADP/NADPH-dependent enzymatic activities were monitored to detect changes induced under the inhibitor stress conditions.  In this study, we report a synthesis procedure for FDM preparation to be used as a standard for HPLC analysis; and an improved ethanologenic yeast strain that can in situ detoxify furfural into FM, and HMF into FDM without the need to allow an initial cell growth phase as in fed-batch cultures prior to exposure to the inhibitors.  Our results suggest that in situ detoxification of furfural and HMF is possible and a mechanism of the detoxification is due to NADH/NADPH-dependent reduction of the functional group aldehyde to the alcohol.