Most insect transmitted pathogens interact with the midgut of their vectors for infection and, in some cases, development. Therefore, molecules associated with the midgut epithelium in direct contact with pathogens may be potential targets of infection-blocking measures. Here, we identify midgut-specific protein antigens from Aedes aegypti (L.) using monoclonal antibodies. Sixty-four monoclonal antibodies were generated with reactivity to the mosquito midgut, five of which are reported in this article. Three monoclonal antibodies identified protein antigens localized at the midgut epithelial surface (the brush border) and the peritrophic membrane. In addition, two monoclonal antibodies recognized mosquito nucleus-specific proteins by immunofluorescence microscopy. Because potential target antigens for blocking transmission of pathogens most likely are located at the interface of mosquito-pathogen interactions, these monoclonal antibodies could provide valuable tools for studying midgut-specific proteins and interactions of the mosquito midgut with pathogens.