Rat liver arylhydroxamic acid N,O-acyltransferase, a noninducible soluble enzyme that can transform N-hydroxy-N-2-aminofluorenes and N-hydroxy-N-acyl-4-aminobiphenyls into reactive derivatives capable of binding protein and oligonucleotides, has been purified greater than 3000-fold by sequential use of the following methods: homogenization and fractional centrifugation, ammonium sulfate precipitation, chromatography on DEAE-cellulose followed by Sephacryl S-200 filtration, preparative polyacrylamide electrophoresis, and preparative isoelectric focusing. These procedures allowed a 14% recovery of enzyme activity. The molecular weight of the enzyme, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 38,500. The isoelectric point, as determined by preparative and analytical flat-bed isoelectrofocusing, is 4.5; the pH optimum is 7.0. N,O-Acyltransferase showed a Km for N-hydroxy-N-acetyl-2-aminofluorene of 6.3 X 10(-6) M with a Vmax of 10.4 nmol of aminofluorene bound to tRNA/min/mg of protein. Activity was not inhibited by the esterase inhibitor paraoxon. Rat liver N,O-acyltransferase is an enzyme that is very unstable, due in part to labile sulfhydryl groups which easily oxidize in air. The enzyme cannot, however, be fully stabilized with the addition of dithiothreitol.