We sought monoclonal antibodies (Mabs) that would recognize distinct subsets of rat tracheal epithelial (RTE) cells. Mice were immunized with pronase-dissociated RTE cells and hybridomas whose supernatants immunocytochemically stained subpopulations of tracheal cells were selected. We report the immunohistochemical staining properties of the antibodies and give the results of preliminary biochemical characterization of the antigens. Four different types of antibodies were produced. Antibody RTE 1 stained most RTE cells. Three antibodies (RTE 2, 7, and 13) recognized a subpopulation of nonciliated cells, both columnar and basal cells. Antibody RTE 3 intensely labeled the surface of ciliated cells. Three antibodies reacted with granule components of secretory cells; antibodies RTE 9 and 11 reacted with mucous-type secretory cells and antibody RTE 12 stained all tracheal surface secretory cells. As described in detail, some antibodies were RTE cell specific while others also reacted with cells and secretions in other organs; the antibodies did not cross react with guinea pig or rabbit tissues. Periodate sensitivity of the antigens suggested that some antibodies recognized carbohydrate moieties while others detected peptide epitopes. In some cases, Western blotting revealed the molecular weights of the antigens, but some antigens were denatured by sodium dodecyl sulfate (SDS) and heat treatment. These antibody probes provide a useful means to immunochemically study changes in cell type distribution and/or epitope expression during development, injury, and regeneration.