The major 28,000- to 36,000-dalton proteins of pulmonary surfactant (SP28-36) have been shown by various techniques to be synthetic and secretory products of alveolar type II cells. Surfactant lipids are also secreted by these cells. Immunocytochemical studies of human and rodent lungs have indicated that nonciliated epithelial cells of small bronchioles appear to contain SP28-36 in their synthetic organelles and secretory granules. Because these observations were obtained with polyclonal antibodies against SP28-36, it was possible that bronchiolar cell staining was due to contaminant antibodies not detected by biochemical analyses. To clarify the role of bronchiolar cells in the metabolism of SP28-36, we have prepared 5 monoclonal antibodies against canine SP28-36. Electrophoresis and immunoblots of surfactant showed that each antibody reacted with SP32 and 36, as well as SP28, the nonglycosylated species. This indicates that the antibodies are directed against the protein rather than carbohydrate moieties of SP28-36. Immunoblot analysis of collagenase-treated SP28-36 showed that the antibodies DS-3 and DS-1 were directed against the noncollagen region of the protein. Immunoblot analysis of whole canine lung homogenates showed that a single protein species was recognized by the antibodies. Immunofluorescence studies of cryostat sections of canine lung showed that both type II and nonciliated bronchiolar cells were specifically labeled with each antibody. These and previous data are consistent with and support the idea that bronchiolar cells synthesize and secrete SP28-36.