B1-type SOXs (SOXs 1, 2, and 3) are the most evolutionarily conserved subgroup of the SOX transcription factor family. To study their maternal functions, we used the affinity-purified antibody antiSOX3c, which inhibits the binding of Xenopus SOX3 to target DNA sequences [Development. 130(2003)5609]. The antibody also cross-reacts with zebrafish embryos. When injected into fertilized Xenopus or zebrafish eggs, antiSOX3c caused a profound gastrulation defect; this defect could be rescued by the injection of RNA encoding SOX3DeltaC-EnR, a SOX3-engrailed repression domain chimera. In antiSOX3c-injected Xenopus embryos, normal animal-vegetal patterning of mesodermal and endodermal markers was disrupted, expression domains were shifted toward the animal pole, and the levels of the endodermal markers SOX17 and endodermin increased. In Xenopus, SOX3 acts as a negative regulator of Xnr5, which encodes a nodal-related TGFbeta-family protein. Two nodal-related proteins are expressed in the early zebrafish embryo, squint and cyclops; antiSOX3c-injection leads to an increase in the level of cyclops expression. In both Xenopus and zebrafish, the antiSOX3c phenotype was rescued by the injection of RNA encoding the nodal inhibitor Cerberus-short (CerS). In Xenopus, antiSOX3c's effects on endodermin expression were suppressed by injection of RNA encoding a dominant negative version of Mixer or a morpholino against SOX17alpha2, both of which act downstream of nodal signaling in the endoderm specification pathway. Based on these data, it appears that maternal B1-type SOX functions together with the VegT/beta-catenin system to regulate nodal expression and to establish the normal pattern of germ layer formation in Xenopus. A mechanistically conserved system appears to act in a similar manner in the zebrafish.