uPA (urokinase-type plasminogen activator) is a potential therapeutic target in a variety of pathological conditions, including cancer. In order to find new principles for inhibiting uPA in murine cancer models, we screened a phage-displayed peptide library with murine uPA as bait. We thereby isolated several murine uPA-binding peptide sequences, the predominant of which was the disulfide-bridged constrained sequence CPAYSRYLDC, which we will refer to as mupain-1. A chemically synthesized peptide corresponding to this sequence was found to be a competitive inhibitor of murine uPA, inhibiting its activity towards a low-molecular-mass chromogenic substrate as well as towards its natural substrate plasminogen. The K(i) value for inhibition as well as the K(D) value for binding were approx. 400 nM. Among a variety of other murine and human serine proteases, including trypsin, mupain-1 was found to be highly selective for murine uPA and did not even measurably inhibit human uPA. The cyclic structure of mupain-1 was indispensable for binding. Alanine scanning mutagenesis identified Arg(6) of mupain-1 as the P1 residue and indicated an extended binding interaction including the P5, P3, P2, P1 and P1' residues of mupain-1 and the specificity pocket, the catalytic triad and amino acids 41, 99 and 192 located in and around the active site of murine uPA. Exchanging His(99) of human uPA by a tyrosine residue, the corresponding residue in murine uPA, conferred mupain-1 susceptibility on to the latter. Peptide-derived inhibitors, such as mupain-1, may provide novel mechanistic information about enzyme-inhibitor interactions, provide alternative methodologies for designing effective protease inhibitors, and be used for target validation in murine model systems.