Formation of the dorsal organizer (Spemann organizer) is an important process in early vertebrate development. In zebrafish, two molecular cascades-Bozozok/Dharma (Boz) and Nodal signaling-act in parallel to induce the dorsal organizer. However, the complete molecular mechanism regulating this event remains unclear. Here we report that zebrafish cell lines derived from various developmental stages can induce a secondary axis when they are implanted into the mid-blastula but not the early gastrula. The implanted cellsthemselves did not differentiate, but instead induced ectopic expression of dorsal organizer markers incells around the implanted cells and induced notochord formation in the secondary axis. These results indicate that cultured cell lines have the ability to induce a secondary axis through the initiation of dorsal organizer activity. However, ectopic expression of boz and sqt were not observed in cultured cells. In addition, implanted cell lines could induce the dorsal organizer even in maternal-zygotic one-eyed pinhead mutants, which are not responsive to Nodal signaling. Finally, the Nodal signaling pathway was not activatedfollowing implantation of cultured cells. Collectively, these data suggest that zebrafish cell lines induce the dorsal organizer independent of the boz and Nodal signaling pathways.