To test the hypothesis that stimulation of glial peroxidase activity by estrogens may play a role in the pathogenesis of the previously reported degenerative changes in the hypothalamic arcuate nucleus that occur in female rats treated with a long-acting estrogen preparation, the cellular localization and number of peroxidase-positive granules in the hypothalamus were determined in adult female rats treated with a single intramuscular injection of 2 mg of estradiol valerate. The persistent estrus state, manifested as persistent vaginal cornification and polycystic ovaries, was induced in 80% of the animals. In comparison with normally cycling controls, the arcuate nuclei of persistent estrus rats exhibited a 3- and 2.8-fold increase in numbers of diaminobenzidine-positive granules and granule clusters, respectively (P less than 0.01 for both comparisons). These peroxidase-positive granules were identified in astrocytes by double-label immunohistochemistry utilizing antiserum to glial fibrillary acidic protein. Diaminobenzidine staining occurred over a pH range of 4-10.5 and was resistant to the catalase inhibitor, aminotriazole, and to tissue pre-heating, indicating that the histochemical reaction was not due to tissue enzyme activity. Rather, these findings indicate that a non-enzymatic, pseudoperoxidation reaction has been induced in these cells by estrogen administration. Possible mediators of this reaction are metallo-porphyrins known to be present in rodent hypothalamus. The mechanisms by which astrocyte peroxidase activity may play a role in estrogen-related neural damage are discussed.