AbstractActivation of gonadotrophin‐releasing hormone (GnRHJ pathways is a pivotal event in the process of sexual maturation, however the regulatory influences that precipitate this change and lead to the onset of puberty remain poorly understood. Recent studies indicate that neuropeptide Y (NPY) may participate in the regulation of luteinizing hormone secretion by modulating the pattern of GnRH secretion and by directly altering the pituitary responsiveness to GnRH stimulation. To determine whether NPY plays a role in puberty‐associated changes in hypothalamic function, levels of NPY‐like immunoreactivity (NPY‐IR) were measured in a fragment of the hypothalamus encompassing the median eminence and medial portion of the arcuate nucleus (ME‐AN), and also in the remainder of the hypothalamus from male rats of different ages. To identify changes in hypothaiamic NPY linked to the process of sexual development, the effect of delaying sexual maturation by daily afternoon administration of 100 μg melatonin (MT) from 20 to 40 days was investigated. In the hypothalamus and ME‐AN, total NPY content increased progressively with age. Expressed as a concentration (fmol/μg extracted protein), peak values for the ME‐AN (55.4 ± 7.0) were observed at 30 days of age followed by a decline to lower levels (30.2 ± 1.9) at 40 days. Daily afternoon administration of MT from 20 days of age resulted in significant increases (P<0.01) in the levels of NPY‐IR in the ME‐AN compared to control values at 30 and 40 days of age. MT was without effect on NPY‐IR levels in the remainder of the hypothalamus. When MT was administered in the early morning, a procedure which does not delay sexual maturation, NPY‐IR values for the ME‐AN region were not different from control rats indicating that the MT‐induced changes in NPY were related to the effects on sexual maturation. Using pituitary luteinizing hormone content and seminal vesicle weight as indices of sexual development, significant inverse correlation coefficients (P<0.001) between these parameters and the NPY concentration in the ME‐AN were observed (r =−0.79 and −0.70, respectively). From published data it is not possible to conclude whether the main effects of NPY are exerted at the hypothalamic or pituitary level. However, the changes in the NPY content of the ME‐AN observed during the onset of puberty, and the influence of MT on these changes, support assertions that NPY is involved i