Human pancreatic adenocarcinoma, an aggressive malignant disease, shows a strong desmoplastic reaction characterized by a remarkable proliferation of interstitial connective tissues. Thrombospondin-1 (TSP-1), a 450 kDa platelet and matrix glycoprotein, has been implicated in tumor invasion, angiogenesis and metastasis. TSP-1 and MMP-9 expression in pancreatic adenocarcinoma and control pancreas tissues was measured by immunohistochemistry. TSP-1 expression in pancreatic carcinoma cell lines, fibroblasts, and endothelial cells was measured by a competitive TSP-1 enzyme linked immunosorbent assay (ELISA). The effect of TSP-1 on MMP-9 production in pancreatic carcinoma cell lines was measured by zymography and Western blot analysis. Eighty five per cent (23/27) of cases of pancreatic adenocarcinoma showed increased TSP-1 staining in the desmoplastic stroma adjacent to tumor cells. No specific positive staining for TSP-1 was observed in the normal pancreatic tissues and the inflammatory areas. TSP-1 localized in tumor stroma surrounding the tumor cells expressing MMP-9. Using TSP-1 competitive ELISA, the secretion of TSP-1 by different pancreatic cancer cell lines into culture medium varied from 11.45 plus minus 14.08 to 275.82 plus minus 45.56 ng/10 6 cells/24 hours. The amounts of TSP-1 detected in both culture media and cell extracts from fibroblasts or endothelial cells were at least 2-3 fold higher than those from pancreatic cancer cells. TSP-1 augmented the production of matrix metalloproteinase-9, a matrix degrading enzyme, in pancreatic cancer cells in vitro. Stromally-derived TSP-1 up-regulates the production of MMP-9 by pancreatic adenocarcinoma. These data are consistent with the conclusion that TSP-1-rich stroma is involved in regulating matrix remodeling in tumor invasion.