We have earlier identified and characterized a tumor-associated trypsin inhibitor (TATI) in the urine of patients with gynecological cancer. Elevated levels of TATI occur in urine and serum of cancer patients (Stenman, U. H., Huhtala, M. L., Koistinen, R. & Seppälä, M. (1982) Int. J. Cancer 30, 53-57). To explain the elevation of TATI in cancer, we have postulated the existence of a tumor-associated protease reacting with TATI. Such a protease, tentatively called protease T, was found in cyst fluid from mucinous ovarian tumors. The protease occurs in complex with TATI, and its protease activity can be measured only after dissociation of the complex. This is achieved by reversed phase chromatography at low pH and elution with an isopropyl alcohol gradient. Protease T is inhibited by phenylmethanesulphonyl fluoride indicating that it is a serine protease. Its optimum activity at pH 9.1 and molecular mass of 24 kDa in gel chromatography are similar to those of trypsin but the substrate specificity is not identical and its isoelectric point (pI) is about 4.0, which is lower than the corresponding values of both cationic (pI 9) and anionic trypsin (pI 5). Protease T could be associated with the elevation of TATI seen in certain tumor patients.