Anorexia and weight loss produced by estradiol (E2) may involve altered expression of neuropeptide Y (NPY) in the hypothalamus. We tested this hypothesis using ovariectomized (OVX) rats by replacing E2 with SILASTIC brand capsule implants and measuring NPY messenger RNA (mRNA) levels in the arcuate nucleus by in situ hybridization. To equalize the effects of weight loss on NPY mRNA expression, E2 deficient OVX rats were pair-fed (n = 10) for 2 days to an OVX group receiving E2 (n = 12). Compared with the weight gain (P < 0.02) of E2 deficient OVX rats (n = 10), OVX rats replaced with E2 and pair-fed OVX rats both had 12.5% lower food intake and weight (P < 0.05). E2 replacement elevated insulin 52% (P < 0.05) and lowered NPY hybridization 32% (P < 0.05) compared with pair-fed controls. During a 2-day fast, E2 replacement (N = 12) attenuated the elevation of NPY mRNA levels 50% (P < 0.01) compared with E2 deficiency (n = 15). Therefore, when E2 is administered to OVX rats, reduced NPY mRNA expression in the hypothalamus is unlikely to be a primary cause of weight loss, although it may contribute to the maintenance of reduced food intake and body weight.