Although transforming growth factor-alpha (TGF-alpha) is widely distributed in transformed cells and in some normal cells and much is known about its structure and metabolism, there is little information about its physiological actions. TGF-alpha is not thought to be synthesized by nontransformed fibroblasts, but it is thought to be a mitogen for these and epithelial cells (Derynck, R. (1986) J. Cell. Biochem. 32, 293-304). We report here that fibroblasts obtained from hamsters with oxidant-induced lung injury release TGF-alpha at levels comparable with those reported for transformed cells. In conditioned media, one isoform of 18 kDa was recognized by a monoclonal antibody to mature TGF-alpha; five isoforms ranging from 18 to 42 kDa were recognized in cell lysates. Conditioned media from these fibroblasts stimulated tyrosine phosphorylation of the epidermal growth factor (EGF)/TGF-alpha receptor, competed with radioactive EGF for binding sites on A431 cells, and were mitogenic for mesenchymal and epithelial cells. This mitogenic activity could be almost completely blocked by anti-TGF-alpha. Conditioned media from normal lung fibroblasts exhibited none of these activities. Using normal lung fibroblasts, we found that TGF-alpha synthesis could be induced in vitro with 25 nmol/ml EGF, suggesting that the induction in vivo may have been due, in part, to a stimulation by EGF (or TGF-alpha) released by other cell types such as alveolar macrophages recruited to the injury site. TGF-alpha is, in general, a mitogen for epithelial cells (Derynck, 1986); more specific to acute injury in the lung, it may affect the proliferation (Ryan, R. M., Mineo-Kuhn, M. M., Kromer, C. M., and Finkelstein, J. N. (1994) Am. J. Physiol. 266, L17-L23) and metabolic activities (Whitsett, J. A., Weaver, T. E., Lieberman, M. A., Clark, J. G., and Daugherty, C. (1987) J. Biol. Chem. 262, 7908-7913) of alveolar epithelial type II cells. This is, we believe, the first report of a fibroblast-derived TGF-alpha induced with oxidant injury. If this response was ubiquitously manifested in other tissues, then fibroblast-derived TGF-alpha might be an important determinant of the epithelial and mesenchymal hyperplasia commonly observed in tissue repair.