Components of the Wnt signaling pathway are involved in patterning the sea urchin primary or animal–vegetal (AV) axis, but the molecular cues that pattern the secondary embryonic axis, the aboral/oral (AO) axis, are not known. In an analysis of signaling molecules that influence patterning along the sea urchin embryonic axes, we found that members of the activin subfamily of transforming growth factor-β (TGF-β) signaling molecules influence the establishment of AO polarities in the early embryo. Injection of activin mRNAs into fertilized eggs or treatment with exogenously applied recombinant activin altered the allocation of ectodermal fates and ventralized the embryo. The phenotypes observed resemble the ventralized phenotype previously reported for NiCl2, a known disrupter of AO patterning. Sensitivity to exogenous activin occurs between fertilization and the late blastula stage, which is also the time of highest NiCl2 sensitivity. These results argue that specification of fates along the embryonic AO axis involves TGF-β signaling. To further examine TGF-β signaling in these embryos, we cloned an endogenous TGF-β from sea urchin embryos that is a member of the activin subfamily, SpNodal, and show through gain of function analysis that it recapitulates results obtained with exogenous activins and NiCl2. The expression pattern of SpNodal is consistent with a role for nodal signaling in the establishment of fates along the AO axis. Loss of function experiments using SpNodal antisense morpholinos also support a role for SpNodal in the establishment of the AO axis. Developmental Dynamics 231:727–740, 2004. © 2004 Wiley-Liss, Inc.