The objectives of these studies were to examine the release of gonadotropin-releasing hormone (GnRH) and β-endorphin-like activity (β-EP) from macaque hypothalami, and the release of luteinizing hormone (LH) and GnRH-induced LH from macaque anterior pituitaries in response to neuropeptide Y (NPY) treatment. Anterior hypothalamic (AH) and mediobasal hypothalamic (MBH) blocks of tissues and the adenohypophysis were bisected along the midline into two equal-sized fragments. Fragments were superfused with medium for 3 h, followed by 3 h of either NPY (80 nM) or medium alone. In a separate experiment, adenohypophyseal (AP) fragments were superfused in accordance with the same protocol (3 h medium – 3 h NPY or medium) except that exogenous GnRH (352 n M) was added for 30 min at the beginning of hour 3 and again at the beginning of hour 6. Immunoactive GnRH, β-EP, and LH levels were measured in superfusate samples (400 µl) collected at 10-min intervals. GnRH levels rose within 20–30 min of initiation of NPY treatment, and elevated GnRH release was sustained for the duration of NPY exposure of both AH and MBH fragments from ovarian intact (INT) rhesus ( Macaca mulatta; n = 8; p < 0.05) or Japanese ( Macaca fascicularis; n = 4; p < 0.01) macaques. NPY treatment had no effect on either AH or MBH fragments isolated from ovariectomized (OVX) rhesus macaques (n = 4 for AH, and n = 5 for MBH). In AP fragments isolated from INT rhesus macaques (n = 8), NPY stimulated LH release within 1 h of treatment (p < 0.05), whereas NPY had no effect on pituitaries from OVX animals (n = 4). Exogenous GnRH stimulated LH release within 20 min; however, the administration of NPY did not alter the responsiveness of Japanese macaque pituitaries to GnRH (p > 0.05; n = 7). NPY treatment had no effect on β-EP release from AH, MBH, and AP tissues of either INT or OVX rhesus macaques. These findings suggest that (1) the stimulatory action of NPY on GnRH release in macaque hypothalami and LH release in macaque anterior pituitaries requires functioning ovaries; (2) NPY does not enhance the sensitivity of macaque gonadotropes to GnRH stimulation, and (3) the mechanism of stimulatory NPY action may not involve the neuronal release of β-EP.