Epidermal growth factor-binding protein (EGF-BP) is a serine proteinase that reversibly associates with epidermal growth factor (EGF). We analyzed the reaction of EGF-BP with urokinase type plasminogen activator (u-PA), a serine proteinase that promotes pericellular proteolysis and cellular migration. EGF-BP cleaved single chain u-PA (scu-PA) between Lys 1 5 8 and Ile 1 5 9 , converting the zymogen into enzymatically active two-chain u-PA (tcu-PA), as shown by SDS-PAGE, N-terminal sequence analysis, and enzymatic assay. The k c a t and K m of the activation reaction were (5.6 ± 0.6) 10 - 2 s - 1 and 2.0 ± 0.3 μM, yielding a catalytic efficiency of 2.8 10 4 M - 1 s - 1 . EGF-BP also activated scu-PA bound to receptors on U937 monocytes as demonstrated by the generation of amidase activity against a tcu-PA-specific fluorogenic substrate. By activating scu-PA, EGF-BP may initiate u-PA-dependent cell surface proteolysis and therefore enhance EGF activities that require cellular migration and/or tissue remodeling.