BACKGROUND.: Cathepsin B, a lysosomal cysteine protease, has a major role in the mechanisms of tumor metastasis. The aim of the present work was to examine the correlation between cathepsin B activity and the metastatic potential of human pancreatic cancer. METHODS.: The primary cell line COLO 357 and the derivative tumor cell lines FG, L3.1, L3.2, L3.3, L3.4, and L3.5, which are characterized by progressively increasing metastatic potential, were injected intrasplenically in the athymic mice. Cathepsin B activity, metastasis, and ultrastructural characteristics were assessed. RESULTS.: An increased number of liver tumor nodules was observed with each subsequent intrasplenic inoculation (p=0.0001), associated with lymph node, splenic, and pancreatic involvement. Cathepsin B activity progressively increased (p=0.001) and was strongly positively correlated with the metastic potential. However, no correlation was found between the metastatic potential and ultrastructural characteristics. CONCLUSIONS.: These findings further support the central role of cathepsin B in metastasis in a combined in vitro/in vivo model.