Nonciliated bronchiolar cells (Clara cells) are thought to provide important respiratory secretions in the small airways and to have other metabolic functions. In mice, nonciliated bronchiolar cells have been the subject of special investigation because tumors of these cells can be specifically induced by chemical carcinogens. A method for isolating nonciliated bronchiolar cells from mice has been reported. However, we have developed a method to isolate these cells from the lungs of BDF1 mice with approximately 80% purity. Cells were identified by electron microscopy, nitroblue tetrazolium dye reduction in the presence of NADPH, immunocytochemical staining of cytochrome P450 isozymes, and mitochondrial staining with rhodamine 123. The isolated cells were examined in culture for synthesis and secretion of proteins and phospholipids. Protein synthesis and secretion were examined in cells labeled with [34S]methionine for 16 h. Fresh medium was added to washed cells and the cells were incubated for an additional 3h. The secreted proteins were precipitated with 10% trichloroacetic acid. Molecular weights of the most prominent radiolabeled secreted proteins were 6, 36, 43, and 45 kDa. Phospholipid synthesis and secretion were examined in cells labeled with [14C]acetate and 32P. Less than 1% of the radioactive lipids was found in the medium, and secretion of lipid was not stimulated by terbutaline or tetradecanoylphorbol acetate compounds, which stimulate phospholipid secretion by type II cells. These data support the hypothesis that nonciliated bronchiolar cells synthesize and secrete proteins but do not secrete phospholipids in any appreciable amount.