In the guinea pig, steroid target cells reside in the ventrolateral hypothalamic nucleus (VLH), an important site in the mediation of female receptive behavior, and in the arcuate nucleus (AR), a structure essential for stimulation effects of ovarian hormones on gonadotropin secretion. However, the mechanisms by which these steroid-dependent reproductive neuroendocrine processes occur are only partially understood. Estrogen is known to affect the hypothalamus content of certain neuropeptides. In the present study, we investigated the effects of estradiol benzoate (EB) on immunoreactivity of neurons containing one of three following neuropeptides: somatostatin (SOM), neurotensin (NT) and substance P (SP) in VLH and AR. The number of immunoreactive (IR)-neurons was quantified in anatomically matched sections through VLH and AR of ovariectomized (OVX), OVX + EB and OVX + oil-treated guinea pigs. Analysis of variance revealed that the number of SOM-IR and SP-IR neurons significantly increased in all regions of VLH of OVX + EB-treated guinea pigs as compared to OVX or OVX + oil-treated animals (P < 0.01) but showed no EB effect on the number of NT-IR neurons. Although the number of SOM-IR and NT-IR neurons slightly increased following treatment with EB in AR, analysis of variance revealed no significant change. The present results provide additional information relevant to possible involvement of these neuropeptides in facilitation of female typical sexual behavior.