To clarify the role of neuropeptide Y (NPY) in the regulation of the reproductive axis, these experiments evaluated the extent to which reproductive hormone secretions may be compromised in the absence of NPY expression. In NPY knockout (NPY-KO) and wild-type (WT) mice, hormone secretions were analyzed under conditions of basal release, following ovariectomy (OVX), in proestrus, after estrogen treatments which induce gonadotropin surges and after injection of gonadotropin-releasing hormone (GnRH). Radioimmunoassays of serum from metestrous females revealed that basal luteinizing hormone (LH), follicular-stimulating hormone (FSH), estrogen and progesterone levels, as well as hypothalamic GnRH tissue concentrations, were not different between the two genotypes. The LH and FSH levels and GnRH tissue concentrations were likewise similar in WT and NPY-KO mice 5 and 10 days following OVX. Significant differences in LH levels were observed however when animals were exposed to pheromone stimulation (male mouse urine) to induce preovulatory LH surges. In proestrous animals, mean LH levels at 18.30–19.00 h were reduced by about 66% in NPY-KO versus WT mice (4.33 ± 1.12 ng/ml in the WT mice vs. 1.47 ± 0.42 ng/ml in the NPY-KO mice, p = 0.028). Despite diminishment of LH surges in NPY-KO mice, corpora lutea were equally abundant in the ovaries of NPY-KO and WT mice. In an additional experiment, a surge-inducing regimen of estradiol-17-β (E<sub>2</sub>) and estradiol benzoate (E<sub>2</sub>B) was administered to OVX animals. The LH surges in the NPY-KO animals treated in this manner were again diminished by approximately 50% compared to corresponding values in WT animals (WT mice 7.33 ± 0.97 ng/ml, NPY-KO mice 3.58 ± 0.74 ng/ml; p = 0.0063). To assess the contribution of altered pituitary responsiveness to the diminishment of LH surges, LH responses to a GnRH challenge (200 ng/kg subcutaneously) were determined; NPY-KO animals exhibited LH responses that were significantly reduced compared to values in WT mice (WT mice 4.88 ± 0.56 ng/ml, NPY-KO mice 3.00 ± 0.41 ng/ml; p = 0.013). Taken together, these observations do not support the idea that NPY plays a major role in the regulation of basal gonadotropin secretion or in mediating negative feedback actions of gonadal hormones. They demonstrate however that preovulatory NPY release is required for normal amplification of the LH surge that occurs on proestrus. Involvement of NPY in the generation of normal LH surges is partially mediated by the ability of the peptide to prime the anterior pituitary gland to GnRH stimulation.