Reproductive maturation and regulation is centrally orchestrated by gonadotropin-releasing hormone (GnRH). GnRH produced in the vertebrate hypothalamus acts on the pituitary to regulate gonadotropins. In nonplacental mammalian species, it has recently been shown that a second GnRH gene is expressed in mesencephalic cells. Here, we report the cDNA sequences and expression patterns for two distinct genes encoding the hypothalamic and mesencephalic GnRH forms in the brain of a placental mammal, the tree shrew (Tupaia glis belangeri). The novel mammalian GnRH form, designated here as [His5Trp7Tyr8]GnRH (often called chicken GnRH II), is expressed in neurons of the mesencephalon and is the first nonhypothalamic form to be isolated from a mammal. Its peptide sequence is identical to the form previously reported in fish, amphibians, reptiles, and birds, revealing that it has remained unchanged for 500 million years. In contrast, the sequences of the hypothalamic GnRH decapeptides vary by as much as 50% across vertebrate species. The remarkable sequence conservation of mesencephalic GnRH suggests that it has been highly constrained throughout evolution, perhaps indicating an important, conserved nongonadotropic role. The discovery and localization of two mRNAs encoding distinct GnRH forms in an advanced mammal suggest that other mammals, including primates, may also have a second GnRH gene with expression localized in the midbrain.