A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW similar to 20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6-10 below 60 A degrees C with an optimum pH of 8.5 at approx. 55 A degrees C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 x 10(3) U (urokinase units) mg(-1) total fibrinolytic activity, which consisted of 692 U mg(-1) direct fibrinolytic activity and 769 U mg(-1) plasminogen-activator activity. K-m and V-max values for azocasein were 1 mg ml(-1) and 43 mu g min(-1) ml(-1), respectively.