We investigated the role of 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) in modulating tumor cell invasiveness through the extracellular matrix (ECM) and pulmonary metastasis in B16 mouse melanoma. The pretreatment of B16 cells for 48 hours with 1alpha,25(OH)2D3 significantly inhibited in vitro invasiveness through the ECM by a mechanism that is not directly correlated with the inhibition of cell proliferation. When cells were treated with 1alpha,25(OH)2D3 for only 8 hours during the assay, no inhibitory effect was observed, suggesting that pretreatment with the hormone for more than 8 hours is necessary to inhibit the invasive potential of B16 cells. The activity of B16 cells to adhere to reconstituted basement membrane (Matrigel) and type IV collagenolysis was inhibited by pretreatment of the cells with 1alpha,25(OH)2D3 for 48 hours. Cell motility was not influenced by the hormone. Mice were inoculated subcutaneously with 3 x 106 B16 cells and were given 1alpha,25(OH)2D3 (0.5 microg/kg) or vehicle daily for 28 days, beginning 1 day after tumor inoculation. In the 1alpha,25(OH)2D3-treated group, no significant inhibition in exponential tumor growth, body weight, and serum level of calcium was observed until the twenty-eighth day. The mean serum concentration of the hormone was about 50 ng/mL, and there were no significant changes in its concentration during the treatment period. In both spontaneous and experimental metastasis models of tumor-bearing mice, treatment with 1alpha,25(OH)2D3 inhibited pulmonary metastasis. These findings suggest that 1alpha,25(OH)2D3 acts on B16 cells, inhibiting invasiveness through the ECM that is caused by the inhibition of cell adhesion to the ECM and the degradation of the ECM by the cells. 1alpha,25(OH)2D3 may have the potential to inhibit metastasis by a mechanism that is not exclusively based on its anti-cell proliferative effect.