Two previously purified esterases of Trichoderma reesei were used to study the deacetylation of polymeric, oligomeric and dimeric acetylated xylan fragments. For the first time nearly complete enzymatic deacetylation of polymeric xylan with purified acetyl xylan esterase was demonstrated, resulting in precipitation of the remaining polymer structure. The esterases had very different substrate specifities, one having a preference for high molecular weight substrates and the other showing high activity only towards acetyl xylobiose. The latter enzyme was also regioselective, cleaving off the acetyl substituent only from the C-3 position of the xylopyranose ring. The highest xylose yield from acetylated xylan was obtained by the synergistic action of xylanase, \-xylosidase and acetyl xylan esterase.