We investigated the effects of various growth inhibitory substances on fibronectin secretion in cultured normal human keratinocytes. Fibronectin secretion in keratinocytes was expressed by two methods: immunofluorescent staining of cells with anti-human fibronectin antibody, and sodium dodecyl-sulphate-polyacrylamide gel electrophoresis of [35S]-labeled proteins secreted by the cells followed by autoradiography. With immunofluorescent staining, the extracellular fibronectin was observed as coarse fibrils only in some cells of the control culture, while cells treated with 20 ng/ml transforming growth factor-beta 1 (TGF-beta 1) showed intense fluorescences in a radiating pattern around most of the cells, indicating that TGF-beta 1 markedly increased fibronectin secretion in keratinocytes. Analysis of [35S]-methionine labeled proteins also revealed that TGF-beta 1 increased fibronectin secretion eight fold in culture medium. TGF-beta 2 has also induced weak increase of fibronectin secretion 1.8 fold in keratinocytes. In contrast, other growth inhibitory substances, such as tumor necrosis factor-alpha, 1,25-dihydroxyvitamin D3 and cyclosporin A, did not show any significant effects on fibronectin secretion. These results indicated that increase of fibronectin secretion is not associated with growth inhibitory effect, but rather with a specific effect of TGF-beta.