Reproductive function is coordinated by the actions of specific neuropeptides and peripheral hormones, converging on the gonadotropin-releasing hormone (GnRH) neurons of the hypothalamic-pituitary-gonadal (HPG) axis. Phoenixin-20 amide (PNX-20) is a recently described peptide that was found to increase GnRH-stimulated LH secretion and GnRH receptor mRNA at the level of the anterior pituitary (1). To date, no studies have looked at the role of PNX-20 at the level of the hypothalamus, where it is most highly expressed. To elucidate the role of PNX-20 at the level of the hypothalamus, immortalized cell lines representative of GnRH and kisspeptin neurons were studied (2). The mHypoA-GnRH/GFP and mHypoA-Kiss/GFP-3 and -4 cell lines represent populations of GnRH, arcuate nucleus kisspeptin and anteroventral periventricular (AVPV) nucleus kisspeptin neurons, respectively. 10 and 100 nM PNX-20 amide treatment in the mHypoA-GnRH/GFP cell line was found to increase c-fos mRNA expression at 30 minutes and GnRH mRNA expression 2-fold after 2 hours. 1000 nM PNX-20 increased the secretion of GnRH after 1 hour and after a 24-hour pre-treatment. 10 nM PNX-20 increased the phosphorylation of ERK1/2. In both the mHypoA-Kiss/GFP-3 and -4 cell lines, 100 nM PNX-20 increased Kiss-1 mRNA by 2-fold at 24 hours. These experiments are the first to implicate a role for PNX-20 at the level of the hypothalamus. PNX-20 appears to stimulate GnRH transcription, protein synthesis and secretion in GnRH neurons and Kiss-1 transcription in both arcuate and AVPV representative kisspeptin neurons. Although a receptor has not yet been described, PNX-20 may use MAPK signal transduction through ERK1/2 to increase GnRH and kisspeptin synthesis. PNX-20 appears to play an important role at the level of the hypothalamus in the regulation of the HPG axis. This work is generously supported by CIHR and CRC.