The study was designed to identify the central pathways through which central interleukin-1β (IL-1β) affects gonadotropin-releasing hormone (GnRH) release in anoestrous ewes. Our results show that intracerebroventricular (icv.) injection of IL-1β (50 μg) decreases the GnRH concentration in the perfusates collected from the medial basal hypothalamus/median eminence (MBH/ME), increases its type I receptor gene expression in the preoptic area, anterior hypothalamus (AHA) and medial basal hypothalamus, and significantly decreases the neuropeptide Y (NPY) mRNA level in the AHA and MBH. No effect of IL-1β treatment was found on plasma cortisol concentration, catecholamine (norepinephrine and dopamine) levels in perfusates, as well as cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) gene expression in the hypothalamus. Our data demonstrate that central IL-1β suppresses GnRH release mainly by acting directly through its own hypothalamic receptors, and its effect could be, at least partially, caused by changes in the NPY level, which is known as an important modulator of GnRH biosynthesis and release.