Proteolytic digestion by larval Diabrotica undecimpunctata howardi (Barber) (D.undecimpunctata) has been investigated with the aim of producing transgenic plants possessing enhanced resistance to this economically important crop pest. Biochemical characterisation in vitro by pH dependent hydrolysis and inhibition assays incorporating E-64, pepstatin A and soybean Kunitz trypsin inhibitor showed the majority of hydrolytic activity occurs at pH 5.5 and is performed by cysteine and aspartic endopeptidases. Cysteine and aspartic proteinase encoding clones were isolated from a larval alimentary tract cDNA library. Four cathepsin L-like cysteine proteinases and two cathepsin D-like aspartic proteinase cDNA clones were identified by codmg homology to known proteinase sequences. Analysis of primary and secondary sequence features revealed D. undecimpunctata aspartic proteinase 1 exhibits features associated with cathepsins E and is proposed to be a D. undecimpunctata cathepsm E-like aspartic proteinase.Cathepsin D-like aspartic proteinase inhibitors of the potato Kunitz protemase inhibitor (PKPI) family have been isolated by PCR and expressed employing the pET expression system (Novagen). In vitro assays demonstrated the inhibitory activity of PKPI-A and PKPl-B inhibitors against larval D. undecimpunctata alimentary tract proteolytic enzymes. To the authors knowledge this work represents the first reporting of the expression and purification of biologically active PKPI proteins. In vitro assays incorporating oryzacystatin I and PKPI proteins resulted in increased inhibition of proteolytic activity compared to single inhibitor and uninhibited control reactions. Inhibition assays provide evidence for the potential of a dual protemase inhibitor strategy to arrest protein hydrolysis by larval D. undecimpunctata, preventing essential amino acid absorption. Further research is necessary to characterise the properties of the digestive enzymes isolated in this work and the inhibitory spectrum of PKPI proteins. Transgenic crops expressing a combination of oryzacystatin and PKPI proteins would be predicted to show enhanced resistance to insect herbivores by virtue of digestive proteolysis inhibition.