This dissertation critically investigates research in melatonin across species and disciplines. It offers a broader perspective within the field of reproductive neuroendocrinology by integrating research in pharmacology, genetics, and evolution. The first chapter of this dissertation reviews melatonin administration techniques across fields. The timing, dose, and mode of administration is compared across species. This juxtaposition of previous protocols using melatonin administration illustrates the importance of context to help inform future experimental designs. Furthermore, details of the experiment that affect the findings are emphasized to improve replicability of melatonin administration studies. The second chapter focuses on melatonin synthesizing enzymes in two different songbirds. The zebra finch (Taeniopygia guttata) is an opportunistic breeder in the wild and uses primarily food and water availability to time reproduction (Perfito et al., 2007). The European starling (Sturnus vulgaris) is a photoperiodic breeder whose reproductive state relies on changes in day length (Gwinner, 1977). Melatonin-synthesizing enzymes were expressed in photoreceptive sites of the brain. Key melatonin-synthesizing enzymes were differentially expressed in the hypothalamus of the photoperiodic breeder at certain times of the year, and this expression was not analogously observed in the opportunistic breeder. Melatonin may be a distinguishing physiological factor in reproductive timing between these two breeding types. The last chapter determines the physiological relevance of melatonin binding at the level of the gonad, specifically measuring how melatonin affects testosterone output from cultured testes from Syrian hamsters (Mesocricetus auratus), a photoperiodically-breeding mammal.