The migration of B16 melanoma cells into porous nitrocellulose filters fitted between the upper and lower compartments of blindwell chemotactic chambers was examined by light microscopy. Human plasma fibronectin placed in the lower compartment of such chambers enhanced in a time-, temperature-, and dose-dependent manner the directed migration of B16 cells into the filter. Fibronectin placed either in the upper compartment alone or in equal concentrations in both compartments did not result in a significant increase in B16 cell migration, indicating that a positive gradient of fibronectin is required. Pretreatment of filters with fibronectin to establish a gradient of bound fibronectin also stimulated the directed migration of B16 cells. The response to fibronectin appeared to be specific, since other plasma proteins and reduced fibronectin or trypsin-digested fibronectin failed to enhance the migration over base-line values. These results suggest that a specific haptotactic-chemotactic response to fibronectin was responsible for enhanced B16 cell migration.