Bone morphogenetic proteins (BMPs) and activins are members of the TGFbeta superfamily of growth factors, a crucial group of regulators of induction and patterning of embryonic germ layers in metazoa. In early Xenopus embryos, activin, Vgl, and nodal are potent inducers of dorsal mesoderm, whereas BMPs can ventralize mesoderm, repress neural fate, and induce blood differentiation. These characteristic responses rely on ligand-specific signaling pathways, encompassing transmembrane kinase receptors and signal transducers belonging to the Smad family. The overexpression in Xenopus embryos of BMP-activated Smad1 and of activin/Vg1/ nodal-activated Smad2 is sufficient to specifically recapitulate ligand responses. In a search for determinants of a Smad specificity code, we have identified two small regions within the conserved carboxyl-domain that are necessary and sufficient for specific Smad action. Swapping both residue clusters (C1 and C2) between Smadl and Smad2 completely switches Smad effects in vivo. Thus, Smadl with swapped Smad2 clusters responds specifically to BMP but elicits an activin response, while a Smad2 protein containing the Smadl clusters is activated by activin and elicits a BMP response. Furthermore, association between Smads and FAST-1, a mediator of mesoderm induction by activin, is dependent upon the presence of the Smad2 C1 sequence. Finally, the Smadl-specific antagonist Smad6 can inhibit a Smad2 molecule harboring Smadl C1 and C2 sequences. Thus, the C1 and C2 regions of Smads specify the association between Smads and pathway-specific partners, such as FAST-1 and Smad6, and account for activin- and BMP- specific responses in vertebrate embryogenesis.