The ability of estradiol-17 alpha to serve as a substrate for estrogen-2/4-hydroxylase in rabbit hypothalamic tissue was determined and compared to that of estradiol-17 beta. Both 2- and 4-hydroxy metabolites of estradiol-17 alpha were formed by the hypothalamic tissue in vitro. The rates of formation of 2-hydroxyestradiol (2-OHE2)-17 alpha and -17 beta were similar as were their kinetic constants (Km and Vmax). In addition, 2-OHE2-17 alpha was shown to inhibit purified rat adrenal tyrosine hydroxylase with a potency comparable to that of 2-OHE2-17 beta, a finding similar to that reported by others with respect to catechol-o-methyltransferase. Since estradiol-17 alpha has a markedly reduced affinity for estrogen receptors compared with extradiol-17 beta, this steroid could be useful in studies designed to distinguish between receptor mediated effects of estrogens and effects that locally formed catechol estrogens may have through their direct interaction with catecholaminergic system in neural tissue.