We recently reported that the steroid hormone, 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) can inhibit growth, alter morphology, and increase cell associated and medium concentrations of fibronectin (FN) in MG-63 human osteosarcoma cells (Franceschi, R. T., James, W., and Zerlauth, G. (1985) J. Cell. Physiol. 123, 401-409). In the present study, we have tested the hypothesis that 1,25-(OH)2D3 increases cellular adhesion by stimulating FN synthesis. Hormone treatment altered cell morphology and increased cell/substratum adhesion in MG-63 cells, effects which could be mimicked by exogenously added FN. 1,25-(OH)2D3-dependent increases in FN production were due to a rapid (within 12 h) increase in FN synthesis. Maximal (2 to 5-fold) stimulation was observed after 48 h. Hormone treatment did not alter apparent FN stability or distribution during this time. The FN response was specific to 1,25-(OH)2D3 when compared with other vitamin D metabolites. In contrast, triamcinolone acetonide, another known inducer of FN synthesis in certain cells, was only slightly stimulatory up to a concentration of 1 microM. FN mRNA, as measured by Northern blot hybridization, increased within 6 h of 1,25-(OH)2D3 addition with maximal (5-fold) induction seen at 24 h. 1,25-(OH)2D3 also stimulated FN synthesis in several other transformed cell lines (TE-85 human osteosarcomas, SW-480 human colon carcinomas, and HL-60 myeloid leukemia cells). These results may be related to known actions of 1,25-(OH)2D3 on cell differentiation and tumor metastasis.