Juvenile hormone esterase (JHE; EC 3.1.1.1), which is intrinsically involved in regulation of development of some insect larvae, is rapidly removed from the hemolymph by the pericardial cells. Lys-29 and Lys-524, which are implicated in the degradation of JHE, were mutated to Arg. Neither the half-life of the modified JHE in the hemolymph nor the catalytic parameters were changed significantly, but when combined, these mutations resulted in apparent failure of lysosomal targeting in the pericardial cell complex. A hypothesis for the mechanism of reduced efficiency of lysosomal targeting is presented. Infection of larvae with a recombinant baculovirus expressing the modified JHE resulted in a 50% reduction in feeding damage compared with larvae infected with the wild-type virus, thus demonstrating improved properties as a biological insecticide. These data demonstrate that alteration of specific residues of JHE that disrupted lysosomal targeting, dramatically increased the insecticidal activity of this protein.