Abstract Acetyl esterases with different specificity occur in one Aspergillus niger preparation. three acetyl esterases were purified and characterised: pectin acetyl esterase (PAE), feruloyl acetyl esterase (FAE) and rhamnogalacturonan acetyl esterase (RGAE). Only PAE, a novel acetyl esterase, could remove acetyl from beet pectin, to a maximum of 30%. This was shown to be one specific acetyl group in the homogalacturonan chain of pectin (smooth region) by NMR spectroscopy. PAE activity was influenced by buffer salts and the addition of bivalent cations. PAE worked cooperatively with pectolytic enzymes. Contrary to PAE, RGAE removed at random the acetyl esters from apple pectic hairy ramified regions (MHR), to a maximum of 70%. RGAE was essential for the activity of rhamnogalacturonase (RG), and as such comparable with A. aculeatus RGAE. FAE was specific for esterified xylan-oligomers, but did not show selectivity towards a specific ester. This enzyme could release ferulic acid as well as acetyl groups from esterified arabinoxylans in the presence of an endoxylanase.