The neuropeptide GnRH-I is critical for the regulation of reproduction in all vertebrates. Study of the regulation of GnRH-I in passerine songbirds has been the focus of studies on subjects as diverse as photoperiodism, puberty, stress, nutrition, processing of auditory information, migration, global climate change, and evolutionary biology. Until now, analysis of GnRH-I in songbirds has been limited to measurement of immunoreactive peptide. Measurement of mRNA regulation has been impossible because of lack of knowledge of the GnRH gene sequence, despite many attempts in the last 20 years to identify it. Thus, the relative roles of environmental, social, physiological, and evolutionary influences upon passerine GnRH regulation have remained enigmatic. Here, we report the first cloning of GnRH-I cDNA from a songbird, Taeniopygia guttata, its localization and regulation. Although the homology of its translated precursor polypeptide between chicken GnRH-I precursor polypeptide was only 54%, zebra finch GnRH-I precursor contained an amino acid sequence that can be processed into chicken GnRH-I peptide (pEHWSYGLQPG-amide). In situ hybridization combined with immunocytochemistry showed co-localization of GnRH-I mRNA and immunoreactive peptide in the preoptic area of sexually mature birds. GnRH-I mRNA signal was greatly reduced in sexually immature birds. Ovary mass of female birds was positively correlated with GnRH-I mRNA level in the brain. These data will now permit molecular analysis of the regulation of songbird reproduction by physical, social, and physiological cues, along with fine scale analysis of selection pressures acting upon the reproductive system of songbirds. (244/250).