Activities of choline acetylase (CAT), synthetic enzyme for acetylcholine, and acetylcholine esterase (AChE), degradative enzyme for acetylcholine, were measured in preoptic nuclei of gonadectomized, estradiol-treated male and female rats. Estradiol-dependent enzymatic changes were localized within discrete POA nuclei and both the location and direction of the changes were dependent on the sex of the rat. Estradiol administration increased CAT and AChE activity in the nucleus of the horizontal limb of the diagonal band in ovariectomized (OVX) females but not castrate (CAS) males and decreased CAT activity in the nucleus of the vertical limb of the diagonal band in CAS males but not OVX females. Activity of AChE was increased by estradiol administration in the bed nucleus of the stria terminalis in OVX females but not in CAS males. Activity of cholinergic enzymes remained higher in the nucleus of the horizontal limb of the diagonal band of females than males after long-term gonadectomy. Estradiol-dependent increased CAT activity in the nucleus of the horizontal limb of the diagonal bands of OVX females was partially blocked by administration of the protein synthesis inhibitor, anisomycin, and was not affected by acute progesterone administration. These results extend previous morphological demonstrations of dimorphisms between the sexes within the preoptic area to include neurochemical parameters of neuronal function.