The microvillar proteome of Manduca sexta larval midguts was analyzed by subjecting brush border membrane vesicles (BBMV) to two different two-dimensional approaches: (i) Anion exchange chromatography followed by SDS-PAGE and (ii) Blue Native-PAGE followed by SDS-PAGE. The first technique was superior to conventional 2-D gel electrophoresis in resolving the most abundant proteins associated with the midgut microvilli. Twenty of them were successfully identified as digestive enzymes, binding targets of the insecticidal Cry1A toxins from Bacillus thuringiensis (Bt), and signal transduction proteins. A homolog of the chlorophyllide A binding protein from the silkworm and several aminopeptidases N represent the most abundant proteins associated with the BBMV. The second technique revealed protein oligomeric complexes associated with midgut microvilli in vivo. Two such complexes contained subunits of the vacuolar ATP synthase complex, and one was an oligomer of the chlorophyllide A binding protein. An additional complex consisted of homo- or hetero-tetramers of three different aminopeptidases N (APNs). As APNs are well-known binding partners of Cry1A toxins, their quaternary structure has implications for Bt toxin mode of action. Both techniques provide a useful complement to conventional 2-D gel electrophoresis in analyzing the complex proteome of the microvillar membrane fraction.