Male testosterone levels decline by 1% per year from the age of 40 years. Whilst a primary testicular deficit occurs, hypothalamic or pituitary dysregulation may also coexist. This study aimed to compare the hypothalamic response to kisspeptin-54 and the pituitary response to gonadotropin-releasing hormone (GnRH) of older men with those of young men. Following 1 h of baseline sampling, healthy older men (n= 5, mean age 59.3±2.9 years) received a 3-h intravenous infusion of either vehicle, kisspeptin-54 0.1, 0.3, or 1.0 nmol/kg/h or GnRH 0.1 nmol/kg/h, on five different study days. Serum gonadotropins and total testosterone were measured every 10 min and compared to those of young men (n= 5/group)(mean age 28.9±2.0 years) with a similar body mass index (24 kg/m 2) who underwent the same protocol. Kisspeptin-54 and GnRH significantly stimulated serum gonadotropin release in older men compared to vehicle (p< 0.001 for all groups). Gonadotropin response to kisspeptin-54 was at least preserved in older men when compared to young men. At the highest dose of kisspeptin-54 (1.0 nmol/kg/h), a significantly greater luteinising hormone (LH)(p= 0.003) response was observed in older men. The follicle-stimulating hormone (FSH) response to GnRH was increased in older men (p= 0.002), but the LH response was similar (p= 0.38). Serum testosterone rises following all doses of kisspeptin-54 (p≤ 0.009) were reduced in older men. Our data suggest that healthy older men without late-onset hypo-gonadism (LOH) have preserved hypothalamic response to kisspeptin-54 and pituitary response to GnRH, but impaired testicular response. Further work is required to investigate the use of kisspeptin-54 to identify hypothalamic deficits in men with LOH.