Juvenile hormone esterase (JHE), which catalyzes the hydrolysis of juvenile hormone, was isolated from the hemolymph of 5(th) instars of Lymantria dispar by two different procedures. One procedure was based on affinity chromatography and the other on anion-exchange chromatography. The material from both purifications showed bands of approximately 50 kDa when analyzed by SDS-PAGE. Isoelectric focusing (IEF) gels in combination with enzyme activity assays indicated two isoelectric forms with the same pI values (pH 5.1. and 5.3) from affinity purification and from anion-exchange chromatography. Amino acid sequencing of several internal peptides from the 50 kDa band following affinity purification and alignment of these sequences with JHEs from previously purified lepidopteran species (Heliothis virescens, Manduca sexta) showed high homology of these enzymes. The isolated JHE, at least in the stage of insect used, was different from the enzyme reported earlier [Valaitis, A.P., 1991. Characterization of hemolymph juvenile hormone esterase from Lymantria dispar. Insect Biochemistry 21, 583-595] to hydrolyze JH in the hemolymph of gypsy moth, based on molecular weight and amino acid sequence.