The distribution pattern of immunoreactive beta-endorphin neurons was studied in female guinea pigs that were ovariectomized, and one week later were injected with 25 micrograms estradiol benzoate or oil. The animals (5 from each group) were perfused after 24 hours with 4% paraformaldehyde. The locations of beta-endorphin cells and fibers were determined using avidin-biotin immunohistochemistry on free-floating vibratome sections. beta-endorphin-immunoreactive fibers were distributed widely throughout specific regions of the rostral forebrain, similar to what has been described in other species. beta-endorphin cell bodies were found in the arcuate nucleus and in adjacent ventrolateral areas throughout the rostrocaudal extent of the basal hypothalamus. Cells immunoreactive to beta-endorphin were also present in the caudal part of the ventromedial nucleus of the hypothalamus. The number of beta-endorphin neurons was quantified in anatomically matched sections through the rostral, medial and caudal basal hypothalamus of estradiol benzoate- and oil-treated guinea pigs. Analysis of variance revealed that the number of immunoreactive beta-endorphin cells was significantly increased in all regions of the basal hypothalamus of estrogen-treated guinea pigs as compared to vehicle-treated animals (P < 0.01). These data indicate that in the guinea pig, the number of neurons expressing beta-endorphin is increased in the arcuate nucleus 24 hours after estrogen treatment.