Technical Abstract: Extensive adoption of Bt cotton places heavy selection pressure on lepidopteran insects. The potential development of Bt resistance is a major risk in the durability of Bt plant technology. Another concern is the surging of non-lepidopteran pests not controlled by Bt toxins. Intensive cloning and sequencing midgut proteinases cDNAs may facilitate future RNAi or other functional study to examine individual role of the proteinases in Bt toxicity and resistance development. This study focused on sequencing of larval midgut proteinase cDNAs from Heliothis virescens and evaluation of proteinase inhibitors for synergistic interaction with Bt toxin, with an ultimate goal to make transgenic cotton versatile and durable. Fifteen chymotrypsin-like cDNAs were cloned and sequenced from the midgut of H. virescens. Most of the predicted chymotrypsins contained the conserved N-termini motif IVGG, three catalytic center residues (His, Asp, and Ser), substrate specificity determinant residue (Ser or Gly), and cysteines for disulfide bridges. These putative chymotrypsins were separated into 3 distinct groups, indicating the diverse proteinases evolved in this polyphagous insect. A sublethal dose (15 ppb) of Cry1Ac, 0.5% benzamidine, and 0.02% phenylmethylsulfonyl fluoride significantly suppressed midgut azocaseinase, tryptic, and chymotryptic activities, and resulted in reductions in larval and pupal body length and mass. The combination of proteinase inhibitor and Bt suppressed approximately 20-37% more larval body mass and 26-80% more enzymatic activities than the inhibitor-only or Bt-only. These results indicated that proteinase inhibitors have potential insecticidal activity and the interaction of Bt with proteinase inhibitors is desirable for enhancing Bt toxicity and delaying resistance development in the target insects.