We investigated the growth-regulatory mechanism of 2 esophageal squamous-cancer cell lines, TE2-NS and TE3-OS cells, both of which can grow stably in protein-free conditions in vitro. Protein-free conditioned media from TE2-NS and TE3-OS cells stimulated the growth of these cells. Exogenous epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), insulin-like growth factor (IGF)-I and -II enhanced cell proliferation by 2.2- to 3.8-fold in protein-free conditions, as compared with an untreated control. Receptor-binding assays showed that both TE2-NS and TE3-OS cells possessed a single class of high-affinity binding sites for IGF-I and 2 classes of binding sites for TGF-α, as confirmed on the cell membrane by immunochemistry. These results suggest that EGF, TGF-α and IGFs are candidates for the autocrine growth factor in cancer cells. The addition of inhibitory monoclonal antibodies against TGF-a and EGFR, but not those against either EGF or IGF-IR, significantly inhibited growth of the cells. Immunocytochemical staining and ELISA of the conditioned media both confirmed the production of TGF-α protein, but not EGF protein, in these cell lines. The data for a protein-free culture system strongly suggested that TGF-α, but not EGF or IGF, is biologically important as an autocrine growth factor in the growth of these cell lines in vitro.