Estrogen-2/4-hydroxylase (E-2/4-H) activity was measured by a direct product isolation assay in punch biopsy specimens obtained from nine nuclear regions from forebrain of adult male rats. Tritiated catechol estrogens were isolated from incubations of tissues with [6,7–3H]estradiol from all regions studied. The amount of 4-hydroxyestradiol (4-OH-E2) formed equaled or exceeded that of 2-hydroxyestradiol (2-OH-E2). There were significant regional differences in the amounts of catechol estrogen produced. The difference was nearly 8-fold between the arcuate-median eminence (ARC-ME) and the medial preoptic nucleus (POM), regions with the highest and lowest specific activities, respectively (37.7 ±6.2 vs. 5.1 ±0.7 pmol/mg protein/10 min 2-OH-E2, mean + SEM, n = 6). The supraoptic nucleus was the site of second highest concentrations of E-2/4-H activity (20.3 pmol 2-OH-E2/mg protein/10 min). Estrogen-2/4-H activity in the paraventricular (PVN) and periventricular (PERI) nuclear regions, though only about half that in the SON, was significantly greater than in the remaining brain areas (nucleus interstitialis striae terminalis, caudate, anterior hypothalamic and medial preoptic nuclei and cortex. The ARC-ME, the region with the highest E-2/4-H activity is where the dopaminergic neurones and terminals from the Gn-RH neurons are concentrated. The functions regulated by these two classes of neurones, the secretion of prolactin and gonadotrophins, respectively, have been the subject of most of the previous studies aimed at establishing the role of catechol estrogen formation in the hypothalamus. The SON and PVN, additional sites with high E-2/4-H activity, are the regions where the magnocellular, oxytocinergic and vasopressinergic neurones are concentrated. These neurones, though recognized targets of estrogens’ action, have not been considered previously potential sites of catechol estrogen formation or as targets of their ac