Gonadal sex determination in vertebrates generally follows a sequence of genetically programmed events. In what is seemingly becoming a pattern, all confirmed or current candidate "master" sex-determining genes reported in this group, e.g., SRY in eutherian mammals, DMY/dmrt1bY in medaka, DM-W in the African clawed frog, and DMRT1 in chicken encode transcription factors. In contrast, here we show that a male-specific, duplicated copy of the anti-Mül-lerian hormone (amh) is implicated in testicular development of the teleost fish Patagonian pejerrey (Odontesthes hatcheri). The gene, termed amhy because it is found in a single metacentric/submetacentric chromosome of XY individuals, is expressed much earlier than the autosomal amh (6 d after fertilization vs. 12 wk after fertilization) and is localized to presumptive Sertoli cells of XY males during testicular differentiation. Moreover, amhy knockdown in XY embryos resulted in the up-regulation of foxl2 and cyp19a1a mRNAs and the development of ovaries. These results are evidence of a functional amh duplication in vertebrates and suggest that amhy may be the master sex-determining gene in this species. If confirmed, this would be a unique instance of a hormone-related gene, a member of the TGF-β superfamily, in such a role.T he sexual fate of the differentiating gonads in vertebrates is under the control of specific genes that initiate and direct the developmental pathway. A few genes have been already identified as master sex determiners, and they all encode transcription factors, e.g., SRY in eutherian mammals (1), DMY/ dmrt1bY in medaka (2, 3), DM-W in the African clawed frog (4), and DMRT1 in chicken (5). These findings might be construed as evidence that transcription factors always trigger gonadal sex determination in vertebrates. However, the molecular pathway of sex determination has been studied in relatively few nonmammalian species, and most of the details of this process remain elusive.We have recently identified a sex-linked locus in Odontesthes hatcheri (Atherinopsidae), a South American gonochoristic fish with an XX-XY sex determination system (6, 7). The existence of a sex-linked single nucleotide polymorphism (SNP) marker associated with this locus has allowed us to profile the expression of a series of genes involved in early sex differentiation of putative females (XX genotype) and males (XY genotype). Analyses performed during early stages of embryonic and larval development revealed a comparatively early mRNA expression of an anti-Müllerian hormone homolog [(amh); also known as Müllerian inhibitory substance/factor, or mis/mif (8)] in relation to other teleosts (9, 10) and showed that this unique feature was due to the up-regulation of a duplicated copy of this gene. AMH, a member of the TGF-β superfamily, is secreted by Sertoli cells and is responsible for the regression of Müllerian ducts during male fetal development in mammals, birds, and reptiles (11-13). Fish have amh even though they lack Müllerian ducts. However, as with mammals and bi...