PURPOSE: Fibroblast activation protein (FAP) is a serine protease that has emerged as a promising target for cancer therapy, either by direct abrogation of its proinvasive activity or by specific targeting of FAP-expressing cells with cytotoxic immunoconjugates. We aimed to select novel human-mouse cross-reactive antibodies and to test suitability for tumor therapy as radioimmunoconjugates in a preclinical model. EXPERIMENTAL DESIGN: Human Fab fragments that bind to human and murine FAP were selected from an antibody phage library. Two candidates (ESC11 and ESC14) were engineered into fully human IgG1 antibodies and further characterized. We investigated the intracellular trafficking of ESC11 and ESC14 in live cells by confocal microscopy and analyzed the biodistribution and therapeutic effects of anti-FAP antibodies labeled with the β-emitting radionuclide (177)Lu in a melanoma xenograft nude mouse model. Results were compared with vF19, a humanized variant of an anti-FAP antibody that has been previously used in clinical trials. RESULTS: The two antibodies bound selectively to both human and mouse FAP, with affinities in the low nanomolar range. Binding to FAP-expressing melanoma cells resulted in rapid internalization of FAP-antibody complexes. (177)Lu-labeled ESC11 specifically accumulated in melanoma xenografts in vivo, with a higher tumor uptake than ESC14 and vF19. Radioimmunotherapy with 8 MBq (177)Lu-labeled anti-FAP antibodies delayed growth of established tumors, whereas (177)Lu-ESC11 extended mouse survival more pronounced than (177)Lu-ESC14 and (177)Lu-vF19. CONCLUSION: Our results show the potential of ESC11 and ESC14 as potent radioimmunoconjugates or antibody-drug conjugates for diagnostic and therapeutic use in patients with FAP-expressing tumors.