Tobacco hornworm, Manduca sexta, is a model insect for studying the action of Bacillus thuringiensis (Bt) Cry1A delta-endotoxins on lepidopterans. The Bt-binding proteins on insect midgut epithelial cells are key factors involved in the insecticidal functions of Bt toxins. With immunohistochemistry, three Cry1A-binding proteins, aminopeptidase N (APN), the cadherin-like Bt-R₁, and membrane-type alkaline phosphatase (m-ALP), were localized on sections from anterior, middle, and posterior regions of midguts from second instar M. sexta larvae Both APN and mALP were distributed predominantly along microvilli in the posterior region and much less so on the apical tip of microvilli in anterior and middle regions. Bt-R₁ was localized at the base of microvilli in the anterior region, over the entire microvilli in the middle region, and at both the apex and base of microvilli in the posterior region. The localization of rhodamine-labeled Cry1Aa, Cry1Ab, and Cry1Ac binding was determined on sections from the same midgut regions. Cry1Aa and Cry1Ab bound to the apical tip of microvilli almost equally in all midgut regions. Binding of Cry1Ac was much stronger in the posterior region than in the anterior and middle ones. With these results we demonstrated that Bt-binding proteins and Cry1A toxins co-localized on the microvilli of M. sexta midgut epithelial cells.