The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3) on cell morphology, the cytoskeleton, and fibronectin were studied in three lines of C3H/10T1/2 mouse embryo fibroblasts in which the antiproliferative effect of the hormone had previously been investigated. We showed that 1,25(OH)2D3 induced morphological changes in the nontransformed C3H/10T1/2 Cl 8 cells, which flattened and spread out markedly. Visualization of actin and tubulin by immunocytochemistry disclosed a reorganization of the microfilament and microtubular systems. 1,25(OH)2D3 also induced an increase in cell-surface-associated fibronectin. These changes were only slight in the transformed cell line C3H/10T1/2 Cl 16 and absent in the transformed C3H/10T1/2 TPA 482 cell line. These effects were correlated with the growth inhibition induced by the hormone, and this suggests a possible relationship between the 1,25(OH)2D3-induced alterations of cell shape and of the cytoskeleton and the effects of the hormone on cell proliferation.