Endopolygalacturonase from Fusarium moniliforme was used to degrade acetylated homogalacturonan previously prepared from sugar beet pulp. The initial velocity and the final percentage of hydrolysis decreased-very rapidly with increasing degree of acetylation, showing that acetyl substitution markedly affected the enzymatic activity. MALDI-TOF mass spectrometry was used to analyse the reaction products and to show acetyl groups on the oligogalacturonates. The results demonstrated that the enzyme was able to accommodate acetyl groups in its active site cleft. The influence of acetyl groups on the mode of action of the enzyme was discussed and compared to the influence of methyl groups. (C) 2003 Elsevier Science Ltd. All rights reserved.