The roles of estrogen in the regulation of pituitary and brain enkephalin systems were studied. A sex-related difference in the levels of enkephalins was observed in the anterior lobe of rat pituitary. Administration of estradiol to male rats caused a marked reduction in the levels of enkephalins in the anterior lobe. Ovariectomy produced a significant increase in the enkephalin levels of the same region but this increase was completely prevented by replacement with estradiol. These results suggest a physiological role of estrogen in the regulation of pituitary enkephalin systems. Repeated injections of haloperidol elicited effects similar to those of estrogen on the pituitary and brain enkephalin levels; namely, striatal enkephalin levels were increased whereas pituitary enkephalin levels were decreased after long-term treatment with these two compounds. These results raise the possibility that the regulation of estrogen on enkephalin systems may in part be mediated through a dopaminergic mechanism.