The homeodomain transcription factor sine oculis-related homeobox 3 (SIX3), is an important regulator of reproduction and circadian rhythms. SIX3 has the potential to be a novel candidate gene for infertility disorders like Idiopathic Hypogonadotropic Hypogonadism (IHH). To extend our knowledge of the importance of Six3, my research investigated the neural location of SIX3 regulation in the hypothalamus that influences fertility and circadian clock. Kisspeptin neurons, in the hypothalamus, are important regulator of endocrine pathways and circadian regulated ovulation. To determine whether SIX3 can regulate the two immortalized kisspeptin neuron populations in vitro, I conducted a transient transfection and luciferase assay. I found that SIX3 is capable of regulating human Kiss1 transcription using human-Kiss1-Luciferase promoter. Another aim of my project was to examine if deletion of Six3 in the neuromedin S (NMS) neurons would affect neuropeptide expression in the suprachiasmatic nucleus (SCN) and/or reproductive competency of male and female mice (SIX3NMSCre). NMS is located in the SCN region of the hypothalamus and an essential pacemaker for internal clock. I prepared samples for in situ hybridization by RNAScope to detect four different types of neuropeptides critical for circadian rhythm, and the data are pending. I also examined male fertility by measuring copulation and sperm in SIX3NMSCre mice. I found that these male mice had normal plugging behavior and total sperm count, however, the percentage of motile sperm is significantly lower in mutant males. To study fertility in SIX3NMSCre female mice, I conducted a fertility assessment which is still underway. Current preliminary results have shown that SIX3NMSCre female mice have normal fertility as measured by time to first litter and number of pups per litter. In conclusion, we found that Six3 expression in kisspeptin neurons is important for female fertility and Six3 expression in the NMS neurons is important for fertility and circadian rhythm in males.