The involvement of central cholinergic mechanisms in the feedback actions of gonadal hormones was examined by measuring choline acetyltransferase activity in rats (ChAT) and acetylcholine concentration (ACh) in microdissected brain nuclei after castration and gonadal hormone replacement. In males, castration resulted in increased plasma luteinizing hormone (LH), as well as in a) increased ChAT in the posteromedial amygdaloid nucleus; b) increased ACh in the ventral tegmental area and rostral nucleus tractus diagonalis; and c) increased ChAT and ACh in the medial preoptic nucleus. Testosterone attenuated all of these responses to castration, and also lowered ChAT in the rostral rostral nucleus tractus diagonalis. Treatment of ovariectomized, estrogen-primed females with progesterone resulted in a surge in plasma LH, as well as in a) increased ChAT in the supraoptic nucleus; b) decreased ChAT in the caudal nucleus tractus diagonalis; c) decreased ACh in the ventral tegmental area; and d) decreased ChAT and ACh in the periventricular nucleus. The results demonstrate that cholinergic activity in several discrete brain regions known to be targets for testicular and ovarian hormones is altered by gonadectomy and gonadal hormone treatment. These brain areas may represent sites where circulating gonadal steroids initiate neuroendocrine feedback actions through cholinergic mediators.