Monoacetylated xylosyl residues of the main hardwood hemicellulose acetylglucuronoxylan undergo acetyl group migration between positions 2 and 3, and predominantly to position 4 of the non-reducing end xylopyranosyl (NRE-Xylp) residues which are amplified by saccharifying enzymes. On monoacetylated non-reducing end xylopyranosyl (NRE-Xylp) residues of xylooligosaccharides the acetyl group migrates predominantly to position 4 and hinders their hydrolysis by beta-xylosidase. Acetyl migration on the NRE-Xylp residues and their enzymatic deacetylation by various xylan deacetylases was followed by H-1-NMR spectroscopy and TLC. A 5-min heat treatment of 4-nitrophenyl 3-O-acetyl-beta-D-xylopyranoside was sufficient to establish equilibrium between monoacetate derivatives acetylated at positions 2, 3 and 4. Rapid acetyl migration along the NRE-Xylp ring at elevated temperature was confirmed in derivatives of methyl beta-1,4-xylotrioside (Xyl(3)Me) monoacetylated solely on the NRE-Xylp residue, the analogues of naturally occurring acetylated xylooligosaccharides. The Xyl(3)Me monoacetates were used as substrates to study regioselectivity of the NRE-Xylp residue deacetylation by various acetylxylan esterases (AcXEs) of distinct carbohydrate esterase (CE) families. CE1, CE4 and CE6 AcXEs hydrolyzed considerably faster the 2"-O-acetyl derivative than the 3"-O-acetyl derivative. In contrast, the CE16 acetyl esterase preferred to attack the ester bond at position 3 followed by position 4. Redistribution of acetyl group on the NRE-Xylp residues is extremely rapid at elevated temperature and includes the formation of 4-acetate. Regioselectivity of AcXEs and CE16 acetyl esterase on these monoacetates is complementary. The formation of all isomers of acetylated xylosyl residues must be taken into account after a long-term incubation of acetylxylan and acetylated xylooligosaccharides solutions or upon their treatment at elevated temperatures. This phenomenon emphasizes requirement of both types of xylan deacetylases to enable a rapid saccharification of xylooligosaccharides by glycoside hydrolases.