We have defined a human lung carcinoma antigen using murine monoclonal antibodies (DF-L1 and DF-L2) prepared against a primary adenocarcinoma of the lung. This antigen is expressed on the surface of human lung carcinoma cell lines and has an apparent Mr of 350,000-420,000. Immunoperoxidase staining has demonstrated expression of the antigen in the cytoplasm and membranes of adenocarcinomas and squamous cell carcinomas but not small cell tumors of the lung. Immunoprecipitation of the antigen following radiolabeling has demonstrated the presence of both protein and carbohydrate. Antigen purified by immunoaffinity was used to study the epitopes defined by monoclonal antibodies DF-L1 and DF-L2. The results indicate that the DF-L1 epitope primarily involves a peptide structure, while the DF-L2 epitope is comprised in part by peptide and O-linked carbohydrate. In contrast, there was no detectable evidence for the presence of N-linked glycosylation. The results also demonstrate that this antigen circulates at elevated levels in patients with carcinoma of the lung. These findings are similar to previous reports of high molecular weight glycoproteins in breast and ovarian carcinomas. Indeed, the present results in lung cancer identify another member of this heterogeneous family of human carcinoma-associated glycoproteins.