Feruloyl esterase and p-coumaroyl esterase activities were separated by ion-exchange chromatography on a column of DEAE-Sepharose CL 6B and then purified by hydrophobic interaction chromatography and anion-exchange chromatography, respectively. Feruloyl esterase had an apparent molecular mass of 112 kDa (SDS-PAGE) and an isoelectric pH of 3.7; the p-coumaroyl esterase was much smaller (75 kDa) and had a pI of 4.2. The feruloyl esterase was highly specific for the methyl ester of ferulic acid and for feruloyl esters associated with a water-soluble xylan polysaccharide from wheat straw; p-coumaroyl esterase was also highly specific for the corresponding p-coumaroyl ester substrates. The wheat straw xylan was de-esterified by the esterases without prior degradation of the polysaccharide; however, esterified short-chain xylooligosaccharides which were generated by a purified fungal xylanase were better substrates. The apparent K m values for the feruloyl and p-coumaroyl residues were 0.93 ± 0.05 and 0.031 ± 0.02 mM, respectively, using the methyl ester substrates; the respective V max values were 2.82 ± 0.05 and 0.57 ± 0.03 μmol min −1 mg −1 protein. The esterases had only a small capacity to release ferulic and p-coumaric acids from cell walls of various plants, including mesophyll cell walls from rye grass, but they were more effective when acting in concert with the commercially available plant cell wall-degrading enzyme Celluclast.