The permeation of macromolecular FITC-labeled dextran (molecular weight 70,000) through bovine aortic endothelial cells (BAEC) monolayer, which were cultured for 5 days with conditioned medium prepared from mouse melanoma B16, was increased. However, when BAEC, which were cultured with normal medium until confluent, were treated with B16 conditioned medium (B16-CM) for 30 min, the permeability did not increase. The B16-CM also increased the permeability of the endothelial monolayers of bovine veins and the human umbilical vein, but did not increase that of the epithelial monolayer. The B16-CM did not alter the distribution or content of F-actin on the BAEC. BAEC cultured in the presence of B16-CM for 5 days were detached from the dish, and then seeded into a chamber at one-fifth of confluent cell density. After 5 days of culture in normal medium, the BAEC were grown to confluence and their permeability was increased. These findings suggest that B16-CM increased the endothelial permeability irreversibly without the decrease of F-actin, and that soluble factor(s) which were secreted from the tumor cells participate in the construction of the hyperpermeable structure of tumor vessels in vivo.