An increased expression of focal adhesion kinase (FAK) in a variety of cancers is associated with a poor disease prognosis. To study the role of FAK in breast tumor growth and metastasis formation, we used conditional doxycycline-regulated expression of a dominant-negative acting splice variant of FAK, FAK-related non-kinase (FRNK), in MTLn3 mammary adenocarcinoma cells in a syngeneic Fischer 344 rat tumor and metastasis model. In cell culture, doxycycline-mediated expression of FRNK inhibited MTLn3 cell spreading and migration in association with reduced formation of focal adhesions and phosphorylation of FAK on Tyr³⁹⁷, but FRNK did not cause apoptosis. Continuous expression of FRNK decreased the primary tumor growth in the mammary fat pad by 60%, which was not due to induction of apoptosis. Lung metastasis formation was almost completely prevented when FRNK was already expressed 1 day before tumor cell injection, whereas expression of FRNK 11 days after injection did not affect lung metastasis formation. FRNK expression during the first 5 days was sufficient to block metastasis formation, excluding the possibility of FRNK-induced dormancy of tumor cells. Together, these data fit with a model wherein FAK is required for breast tumor cell invasion/migration processes that take place in the early phase of metastasis formation. Our findings suggest that FAK is a good candidate for therapeutic intervention of metastasis formation.