Previous studies have indicated that cyst fluid of ovarian tumors contains 2 trypsinogen isoenzymes, called tumor-associated trypsinogen-I (TAT-I) and trypsinogen-2 (TAT-2), the levels of which correlate with the degree of malignancy of the tumors. In addition, these cyst fluids contain large amounts of tumor-associated trypsin inhibitor (TATI), which is also expressed in many other human tumors. In the present study we examined the production of TAT-I, TAT-2 and TATI in 9 established tumor-cell lines. TAT-2 was produced by 5 cell lines. Its concentration in the conditioned medium of COLO 205 colon adenocarcinoma cells, K-562 erythroleukemia cells and fibrosarcoma cell lines HT 1080, 8387 and A 9733 was 460 micrograms/l, 9.8 micrograms/l, 21 micrograms/l, 8.8 micrograms/l and 0.24 micrograms/l, respectively. TAT-I was detectable in the conditioned medium of COLO 205 and HT 1080 cells at concentrations of 64 micrograms/l and 0.5 micrograms/l, respectively. TATI was detected only in the media of COLO 205 cells at a concentration of 23 micrograms/l. TAT-2 zymogen was purified from the conditioned medium of COLO 205 and HT 1080 cells by immunoaffinity chromatography. According to its aminoterminal amino acid sequence, a molecular mass of 28 kDa by SDS-PAGE, elution pattern in ion-exchange chromatography and ability to be activated by enteropeptidase, the zymogen is identical to that previously isolated from cyst fluid of ovarian tumors. In addition, we found that TAT-2 secretion could be down-regulated by dexamethasone in HT 1080 cells but not in COLO 205 cells. The abundant production of TAT-2 isoenzyme in different cancer cell lines suggests that it could contribute to the increased proteolytic activity of many human tumors.