A panel of murine monoclonal antibodies was generated against a high-molecular-weight glycoprotein produced by human lung cancer cells. This lung cancer-associated protein (LCAP) has been shown to circulate in the plasma of patients with lung cancer. Various combinations of MAbs were used in solid-phase enzyme-linked sandwich immunoassays to optimize the detection of LCAP in the plasma of these patients. One of these monoclonal antibodies, designated DF-L1, used both in the solid phase as well as the tracer, was selected to evaluate circulating levels of LCAP in normal subjects and in patients with lung cancer. In 341 normal subjects, the mean LCAP level was 7 units/ml, with 47 (13.8%) and 18 (5.3%) subjects having levels greater than or equal to 15 units/ml and 23 units/ml, respectively. In contrast, 27 of 35 (77.1%) patients with lung cancer had LCAP levels greater than or equal to 23 units/ml. A total of 16 of 19 (84.2%) patients with adenocarcinoma, four of seven (57.1%) patients with squamous cell carcinoma, and four of six (66.7%) patients with small cell carcinoma had levels greater than or equal to 23 units/ml. Moreover, in a small group of patients, serial LCAP levels correlated with clinical course during therapy. The LCAP assay is technically reproducible and unaffected by interfering substances in the blood or by variations in the handling of samples. These results indicate that LCAP is a new and potentially useful marker for the evaluation of patients with lung cancer.