Single- and double-immunohistochemical staining methods were used to assay the effect of estrogen on the expression of co-existing peptides in the magnocellular neurosecretory system of the female rat. It was confirmed in colchicine-treated, ovariectomized animals that immunoreactive corticotropin-releasing factor and cholecystokinin co-exist in subsets of oxytocinergic neurons; in addition, dynorphin immunoreactivity was detected in a substantial majority of oxytocin containing magnocellular neurons. Consistent with previous studies, magnocellular vasopressinergic cells were found to display angiotensin II-, dynorphin- and galanin-immunoreactivities. Comparable results occurred in colchicine-treated ovariectomized rats independent of whether or not the animals received replacement injections of estradiol benzoate or vehicle. Ovariectomized rats that were not pretreated with colchicine showed enhanced staining (increased cell number and staining intensity of both cell bodies and terminals in the posterior pituitary) for each of the peptides that was found to co-exist in vasopressinergic neurons after treatment with estradiol; staining for vasopressin was similar in steroid- and oil-treated animals. Perikaryal staining for peptides co-localized with oxytocin was not discernibly different in estradiol- vs vehicle-treated animals, while in the posterior lobe, differential effects of hormone replacement on oxytocin, cholecystokinin, and corticotropin-releasing factor immunostaining of terminals were apparent. Perikaryal staining for co-existing peptides in gonadally intact animals killed at the estrus or the diestrus II phases of the estrous cycle provided a pattern of results compatible with those seen in ovariectomized animals treated with estradiol or oil, respectively. These observations suggest that circulating gonadal steroids affect co-existing peptide expression differentially in oxytocinergic vs vasopressinergic neurons. All peptides examined that could be co-localized in vasopressinergic cells showed evidence of enhanced expression in the presence of estrogen, while at least two of these co-localized with oxytocin appeared driven in the opposite direction. The results in normally cycling rats indicate that this kind of influence may be manifest under normal physiological conditions.