The distribution of acetyl esterase was studied in 30 strains of wood-rotting fungi. A screening test on agar plates using glucose β-d-pentaacetate as a substrate indicated that all tested fungi produced acetyl esterase to form a clear zone on the culture. All fungi also showed positive responses in an agar test using carboxymethyl cellulose acetate. Enzyme assay showed that extracellular acetylxylan esterase activity was present in the filtrates of wood-meal culture of all these fungi. The ratio of fungal acetylxylan esterase activity to 4-nitrophenyl acetyl esterase activity were higher than that of porcine liver esterase, indicating that fungal esterases have high affinity for acetylated carbohydrates. Acetyl esterase is suggested to be distributed widely in wood-rotting fungi for degradation of native acetylated hemicelluloses.