The insecticidal efficacy of baculoviruses can be enhanced by engineering the viral genome to express proteins that disrupt the physiology of the host insect. Here we describe the development of a genetically engineered Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) which expresses a modified form of juvenile hormone esterase (JHE). Previously, two viruses expressing different modified JHEs were found to have a greater insecticidal effect on larvae of Trichoplusia ni and Heliothis virescens than a virus expressing wild-type JHE. To study a possible synergistic effect, the distinct mutations in the modified JHEs were combined in a new JHE construct. Two lysine residues were replaced with arginine residues to reduce the efficiency of lysosomal targeting (JHE-KK) and the catalytic serine was replaced with glycine, which eliminated catalytic activity (JHE-SG). The modified JHE, JHE-KSK, was expressed in a recombinant baculovirus, AcJHE-KSK. Larvae of H. virescens infected with this recombinant virus caused 44␕ess feeding damage to lettuce than larvae infected with the wild-type AcMNPV. However, AcJHE-KSK did not have significantly improved insecticidal properties over the parent viruses AcJHE-KK and AcJHE-SG, suggesting that the separate mutations have no major synergistic effect. Infection with a control recombinant baculovirus expressing JHE with the same lysine to arginine conversions and in which a catalytic histidine was converted to lysine (AcJHE-KHK) did not reduce feeding damage compared with that caused by larvae infected with AcMNPV.