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Two cellulose acetates (CA) were regioselectively deacetylated by action of a pure Aspergillus niger acetylesterase from the carbohydrate esterase family 1. The action of acetyl esterase along the polymeric chain was monitored by a new enzyme-aided method. CA with and without esterase modification was hydrolysed with a pure endoglucanase. The fragments were deutero-acetylated and separated by preparative SEC in CHCl3. The partial degree of substitution of the individual fragments was determined by 1H-NMR spectroscopy. The investigation confirmed the uniform regioselective and regular deacetylation along the polysaccharide chain. The partial substitution in C2 seemed to be of major importance for the enzyme's mode of action.