Dilute-acid biomass hydrolysates contain biomass degradation products that are inhibitory to cell growth and fermentation. Overliming with Ca(OH)(2) has been found to be one of the most effective methods for detoxifying the dilute-acid hydrolysate for ethanol production. However, the mechanism of overliming is not well understood. Carbon-13 nuclear magnetic resonance (C-13-NMR) spectroscopy was used to elucidate the functional groups involved in the overliming reaction. The C-13-NMR spectra showed that the major functional groups removed during the overliming process were aliphatic and aromatic acids or esters, and other aromatic and aliphatic compounds. Ketone and aldehyde functionalities were not detected in the spectra. This is the first time that C-13-NMR has been used to elucidate the overliming reaction.