The lysosomal cysteine proteinase cathepsin L is involved in proteolytic processing of internalized proteins. In transformed cells, where it is frequently overexpressed, its intracellular localization and functions can be altered. Previously, we reported that treatment of highly metastatic, murine carcinoma H-59 cells with small molecule cysteine proteinase inhibitors altered the responsiveness of the type I insulin-like growth factor (IGF-I) receptor and consequently reduced cell invasion and metastasis. To assess more specifically the role of cathepsin L in IGF-I-induced signaling and tumorigenicity, we generated H-59 subclones with reduced cathepsin L expression levels. These clonal lines showed an altered responsiveness to IGF-I in vitro, as evidenced by (i) loss of IGF-I-induced receptor phosphorylation and Shc recruitment, (ii) reduced IGF-I (but not IGF-II)-induced cellular proliferation and migration, (iii) decreased anchorage-independent growth and (iv) reduced plasma membrane levels of IGF-IR. These changes resulted in increased apoptosis in vivo and an impaired ability of the cells to form liver metastases. The results demonstrate that cathepsin L expression levels regulate cell responsiveness to IGF-I and thereby identify a novel function for cathepsin L in the control of the tumorigenic/metastatic phenotype.