The urokinase plasminogen activator (uPA) is implicated in both cancer cell invasion and angiogenesis. It can interact with a specific receptor (uPAR) via the epidermal growth factor (EGF)-like domain in the urokinase amino-terminal fragment (ATF) in a species-specific manner. Our previous studies showed that adenovirusmediated delivery of murine ATF (AdmATF) suppressed human tumor growth in mouse models, by inhibiting murine angiogenesis. However, we cannot exclude its putative inhibitory action on human cancer cell invasion through a uPAR-independent pathway. To further investigate the mechanisms of ATF, we constructed another adenovirus, AdhmATF, expressing humanized murine ATF (hmATF). hmATF binds to human uPAR but not to murine uPAR. We compared the antagonist effect of both AdmATF and AdhmATF on human and murine cancer cells. In vitro, the supernatant from AdhmATF-infected cells repressed 79% of membrane-associated uPA activity on human MDA-MB-231 cells, whereas that from AdmATF-infected cells repressed 35% of membrane-associated uPA activity. On murine LLC cells, the supernatant from AdhmATF-infected cells inhibited 29% of cell surface uPA activity, whereas that from AdmATF-infected cells inhibited 74% of cell surface uPA activity. Similar results were obtained in a cell invasion assay. In vivo, intratumoral injection of the adenoviruses into LLC tumors on day 24 postinjection induced lower but significant tumor growth suppression by AdhmATF (tumor volume was 1185 +/- 128 mm3), whereas suppression by AdmATF was greater (407 +/- 147 mm3). In the MDA-MB-231 tumor model, on day 52 postinjection, tumor size was 187 +/- 47 mm3 in the AdhmATF-treated group and 468 +/- 65 mm3 in the AdmATF-treated group. The LLC and MDA-MB- 231 cell lines transfected by mATF or hmATF genes showed growth inhibition In vivo equivalent to the results obtained by adenovirus treatment. These results demonstrate the strong anticancer activity of ATF even when its uPAR-binding affinity has been suppressed, and indicate that ATF exerts an antitumor effect via dual mechanisms: essentially through targeting the uPA-uPAR system via the EGF-like domain and partially through targeting a uPAR-independent interaction via the kringle domain.