Thrombomodulin (TM) is an anticoagulant molecule expressed on the endothelial cell surface and soluble TM antigen, which is present in human plasma and urine, represents the products of limited proteolytic cleavage of cell-surface TM. Recently, it was demonstrated that TM is also expressed on the surface of several tumor cells and the expression level of TM negatively correlated with malignancy in cancer. We investigated the effect of soluble TM isolated from human urine (uTM) on the invasion and metastasis of murine melanoma cells (B16F10 cells) through a reconstituted basement membrane (Matrigel) and in a murine model of experimental lung metastasis. Matrigel reconstituted with uTM inhibited the invasion of B16F10 cells in a dose-dependent manner in a range from 10 to 1000 ng/ml uTM as compared with the control Matrigel without uTM. The inhibitory action of uTM was not altered in the presence of an excess amount of hirudin, an inhibitor of thrombin proteolytic activity, but abolished in the presence of anti-human TM IgG. Matrigel reconstituted with thrombin (1 NIH unit/ml) enhanced the invasion level of cells by 1.5-fold relative to the control Matrigel without thrombin. The thrombin-enhanced invasion of B16F10 cells was repressed by addition of hirudin (10 units/ml) or uTM (100 ng/ml) into the Matrigel. Matrigel reconstituted with hirudin (10 units/ml) and uTM (100 ng/ml) additionally accelerated the inhibitory activity of hirudin or uTM on the thrombin-enhanced invasion of B16F10 cells. Moreover, metastatic colonies formed in the lungs of mice injected intravenously with B16F10 cells were significantly reduced by injection of uTM once a day up to 2 days after co-injection of uTM with the cells. These results suggested that Matrigel reconstituted with uTM inhibited the invasion of B16F10 cells in vitro through a thrombin-independent mechanism and the injection of uTM suppressed experimental lung metastasis of the cells in mice.