In order to assess the possible involvement of central cholinergic mechanisms in the feedback actions of gonadal hormones, the activity of choline acetyltransferase (ChAT) and the concentration of acetylcholine (ACh) were measured in microdissected brain nuclei of male and female rats after castration and gonadal hormone replacement. Castration of male rats significantly elevated plasma levels of LH and also increased ChAT activity and ACh level in the medial preoptic nucleus. Castration also increased ChAT activity but not ACh concentration in the posteromedial amygdala. In the rostral part of the nucleus tractus diagonalis and in the ventral tegmental area. ACh concentrations were elevated by castration, but ChAT activity was unaffected. Daily administration of testosterone propionate (TP) to castrated males attenuated the postcastration rise of plasma LH and also partially prevented the increases of ACh in the medial preoptic and rostral tractus diagonalis nuclei and of ChAT in the posteromedial amygdala. In addition, TP treatment significantly decreased ChAT acivity in the rostral nucleus tractus diagonalis. Treatment of ovariectomized female rats with estradiol benzoate lowered plasma levels of LH, but did not affect cholinergic parameters. Administration of progesterone to estrogen-primed females produced a surge in plasma LH and decreased the activity of ChAT and the concentration of ACh in the periventricular nucleus. Such treatment also reduced ChAT activity in the caudal nucleus tractus diagonalis and decreased ACh levels in the ventral tegmental area. The combined estradiol/progesterone treatment elevated ChAT activity in the supraoptic nucleus. These 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 and suggest involvement of cholinergic systems in the feedback effects of estadiol, progesterone and testosterone.