Biopsy tissue from a 72-year-old Japanese woman with metastasis to the cervical lymph nodes of recurrent esophageal carcinoma was adapted for culturing on the surface of lethally X-irradiated confluent monolayers of Balb/c 3T3 cells, and a continuous-growing tumor cell line was developed. These cells were found to contain keratinous material by immunohistochemical staining. We confirmed that these cells were of human origin by performing chromosome analysis. Thus, this line, designated as KSE-2 line, was considered to have originated from metastatic squamous-cell carcinoma of the human esophagus. When the attachment and growth of KSE-2 was examined on various culture surfaces, the confluent monolayers of Balb/c 3T3 cells were found to be more suitable than other substrata, such as plastic and collagen-coated dishes. The fixation of confluent monolayers of 3T3 cells with 3% glutaraldehyde had almost no affect on the colony-forming efficiency of the KSE-2 cells. In addition, the period of lag phase after inoculation of KSE-2 cells on fixed as well as nonfixed monolayers was shorter than that on plastic dishes. The conditioned medium from lethally X-irradiated 3T3 cells did not enhance the attachment and proliferation of KSE-2 cells on both nonfixed and fixed confluent monolayers. From these results, the surface structure itself of the 3T3 confluent monolayer was considered to play an important role in the favorable attachment and proliferation of KSE-2 cells.