During vertebrate embryogenesis, blastoderm cells at the gastrula stage migrate to new locations for subsequent development. The cellular mechanism of migration was studied in medaka (Oryzias latipes) embryos at the early gastrula stage. When fibronectin was applied iontophoretically or by the puff method, cell surface protrusion known as pseudopods and a local [Ca²⁺]_i rise at the site of application were observed in approximately half of the isolated blastoderm cells. When the pseudopod adhered to the substrate, the cell body moved toward the direction of the pseudopod as [Ca²⁺]_i declined and the pseudopod was withdrawn. Local puff application of ionomycin, a Ca²⁺ ionophore, in the presence of external Ca²⁺ induced protrusions of the plasma membrane similar to pseudopods, suggesting that the [Ca²⁺]_i rise itself is causing pseudopod formation. On the other hand, fibronectin induced pseudopods even in the absence of external Ca²⁺, suggesting the mobilization of Ca²⁺ from internal stores. In accordance with this interpretation, fibronectin failed to induce [Ca²⁺]_i rises after pretreatment with thapsigargin, a blocker of Ca²⁺-ATPase in the endoplasmic reticulum. Furthermore, chelating internal Ca²⁺ with BAPTA prevented fibronectin from inducing pseudopods. U-73122, a blocker of phospholipase C, completely suppressed both the [Ca²⁺]_i rise and morphological changes accompanied with fibronectin application, suggesting involvement of the inositol phosphate pathway. On the other hand, caffeine evoked a [Ca²⁺]_i rise in a great majority of the fibronectin-responsive cells and the percentage of fibronectin-responsive cells was greatly reduced by a blocking dose of ryanodine. These results suggest that fibronectin activates phospholipase C and the initial [Ca²⁺]_i rise through IP₃ receptors further activates ryanodine receptors, achieving the local [Ca²⁺]_i rise. The decay time course of [Ca²⁺]_i after fibronectin application was prolonged in the absence of external Na⁺. DCB, an inhibitor of Na⁺/Ca²⁺ exchangers, also prolonged the time course of the [Ca²⁺]_i decay, suggesting the contribution of Na⁺/Ca²⁺ exchangers. Cytochalasin D, an inhibitor of actin polymerization by binding to the barbed end of F-actin, induced swelling in fibronectin-responsive cells and prevented fibronectin from inducing pseudopod formation without suppressing the [Ca²⁺]_i rise. These results support the hypothesis that fibronectin facilitates cell migration via pseudopod formation during gastrulation.