Single-chain pro-urokinase (pro-uPA) is present both in the medium and lysate of the A431 epidermoid carcinoma cell line. Most of the cell-associated pro-uPA is on the cell surface, as shown by indirect immunofluorescence and by surface iodination. Pro-uPA is not an integral membrane protein but is bound to a specific surface receptor that is completely saturated. A mild acid treatment uncovers the surface receptors by dissociating pro-uPA. Resaturation of uncovered receptors has been studied by reincubating cells in normal medium; within 40 min, 50% of the free sites are reoccupied. Excess uPA-specific antibodies prevent rebinding of ligand to the receptors. Thus, A431 cells first secrete uPA, which then binds to the surface receptor. We propose that the synthesis of uPA and uPA receptor by the same cell may provide a pathway for the activation of the metastatic potential of malignant cells.