Mammalian urokinase-type plasminogen activator (uPA) is produced as a stable single polypeptide chain zymogen and requires a distinct proteolytic cleavage to become an active, two-chain enzyme. In contrast, chicken uPA, both native and recombinant, is found predominantly as a two-chain, active enzyme even in the absence of plasmin, a physiological activator. Here we show that the proclivity to autoactivate is not a unique property of the chicken uPA catalytic domain but requires sequences distinct from and independent of the serine protease domain. Human/chicken chimeric uPA molecules and point mutants were used to determine the structural requirements for uPA autoactivation versus zymogen stability. The amino terminal fragment of chicken uPA engineered onto the human uPA molecule can induce the autoactivation of the human uPA. In fact, the first twenty residues of the chicken uPA are necessary and sufficient to induce the autoactivation of chicken and human uPA. These results indicate that sequence motifs, distal to the active site, control the substrate specificity and catalytic efficiency of uPA activity in autolytic activation.