Arylacetamide deacetylation is an important enzyme activity in the metabolic activation of arylamine substrates to ultimate carcinogens, best described as a carboxylesterase/amidase type of reaction. A 7-fold variation in the Vmax of 2-acetylaminofluorene deacetylation in 24 human livers was observed. An acetylaminofluorene deacetylase was purified 90 fold from human liver microsomes by PEG-fractionation, anion exchange and hydrophobic interaction chromatography. The purified 45kD protein showed no amino acid sequence homology to other carboxylesterases, neither in its N-terminus nor in tryptic peptides. Antibodies raised against the deacetylase recognized the protein with high specificity. This report thus describes the first arylacetamide deacetylase in human liver.