Forty-one human pituitary adenoma specimens were examined for the presence of estrogen receptor (ER) messenger ribonucleic acid and protein using a combination of ribonuclease protection assay, [3H] estradiol ([3H]E2) binding, and ER immunohistochemistry. ER messenger ribonucleic acid prevalence was high in PRL-immunoreactive tumors (2 of 2), moderate in GH/PRL tumors (2 of 5), and low or absent (0 of 4) in GH tumors. In the GH/PRL-immunostaining tumors, the presence of the ER was uniformly associated with elevated serum PRL levels. Among the gonadotropin-immunostaining tumors, 10 of 17 were ER positive; within this group, those with gonadotroph adenoma characteristics were ER positive, whereas those with null cell/oncocytic characteristics were ER negative. Of the tumors that did not immunostain for any known anterior pituitary hormones, 3 of 11 were ER positive. ER immunohistochemistry in 14 tumors revealed a 100% correlation with ribonuclease protection assay results, whereas [3H]E2 binding, determined in 9 tumors, showed an 87% correlation. In summary, it appears that PRL and a specific class of gonadotropin-immunostaining tumors (identifiable by specific characteristics on electron microscope) contain ER, whereas GH-immunostaining tumors are ER negative. ER expression in normal pituitary paralleled that in macroadenomas (GH, 2.3%; PRL, 50%; FSH, 70%; LH, 83%; TSH, 4%; ACTH, 1%). The ER-positive tumors represent a subset whose growth and secretory profiles may be influenced by the gonadal steroidal milieu or by pharmacological agents that affect E2 levels or ER function.