To examine whether a human carcinoembryonic antigen (CEA)-producing cell can proliferate and sythesize CEA in vitro in culture without protein supplements, long-term cultivation of such cells was carried out in a protein-free chemically defined medium. Using stepwise decreases in fetal bovine serum concentration, continuous growth of the cells was established in a protein-free am's F-12 medium. The cells, designated as HLC-Yl, have been propagated in this medium for 3 years. The population doubling time of the cells is about 52 h. Addition of the serum stimulated the cell growth (population doubling time = 27 h). Saturation density was not increased by the addition of serum. The cells grown in a protein-free F-12 secrete large amounts of CEA (65.4 +/- 2.6 ng/10(6) cells/24 h). Addition of serum did not stimulate the production of CEA. The cells produced tumours when inoculated into athymic nude mice. The mice bearing the tumour showed high serum CEA levels, and CEA was demonstrated in the tumour tissue by the immunoperoxidase method. The present study suggests that cells grown in a protein-free medium do not require serum components for their growth or CEA synthesis and provide an excellent model for better understanding the growth and production of CEA in human lung cancer cells.