An Expressed Sequence Tag(EST) analysis of the gut tissue of larvae of the yellow mealworm, Tenebrio molitor, was completed as a first step in the study of proteinase gene regulation at the transcriptional and translational level as well as the response of T. molitor larvae to proteinase inhibitors. Of 1,528 cDNA sequences, 92 sequences encoded potential proteinases from the midgut of T. molitor including many full-length cDNA sequences. The remaining non-proteinase cDNA sequences were analyzed by their homology to known genes. Other sequences included enzymes associated with digestion, as well as those responsible for maintenance of basic gut physiology, such as the peritrophic membrane and energy metabolism. Proteinase sequences were grouped into functional classes and further separated into clusters. Results indicated representative proteinase genes from the serine, cysteine and metalloproteinase class and are consistent with previous biochemical studies from the T. molitor midgut. Specific proteinases included chymotrypsin, trypsin, elastase, and cathepsin B and L. The presence of these transcripts was confirmed by northern blot analysis, and real-time PCR was used to quantify and evaluate temporal expression of representative proteinase genes in different developmental stages of T. molitor.