The zebrafish wnt8 locus differs from its tetrapod counterparts in that it produces two functionally overlapping but distinct Wnt8 proteins. Studies of zebrafish wnt8 have suggested that the two major Wnt8 proteins produced are functionally similar yet may behave differently depending on the assay context. To determine whether the bicistronic wnt8 and its accompanying unique protein activities found in zebrafish are more widespread (and perhaps universal) among teleosts, we have extended our studies to the pufferfish Takifugu rubripes. We have found that Takifugu wnt8 is also bicistronic, indicating that the wnt8 duplication occurred before the divergence of these teleosts approximately 150 million years ago. Furthermore, overexpression assays in zebrafish embryos show that functional differences between the zebrafish Wnt8.1 and Wnt8.2 proteins are conserved in their Takifugu orthologs. Thus, despite the fact that Wnt8.1 and Wnt8.2 proteins are as similar to each other as each is to Xenopus Xwnt-8, Wnt8 family members can behave quite differently in the context of zebrafish embryos. This finding suggests that zebrafish (and possibly teleost in general) Wnt8 receptors are able to discriminate between highly related ligands.