The Western Corn rootworm, Diabrotica virgifera virgifera is the major pest of corn in the USA and has recently become the target for insect resistant transgenic crops encoding Bt toxins. Transgenic crops have switched the focus for identifying insecticide targets from the insect nervous system to the midgut. Here we describe a collection of 691 sequences from the D. v. virgifera midgut. These sequences correspond to 691 unique nucleotide sequences, 27% of which predict proteins with no matches in current databases. Of the remaining sequences, most predict proteins with either catalytic (62%) or binding (19%) functions, as expected for proteins expressed in the insect midgut. The utility of this approach for the identification of targets for novel toxins is demonstrated by analysis of the first coleopteran cadherin gene, a putative Bt receptor, and a large class of cysteine-proteases, the cathepsins. A number of carboxylesterase genes were also identified which may confer resistance to organosphosphate insecticides.