A 43-yr-old woman, who had previously had a subtotal thyroidectomy, presented with hyperthyroidism and amenorrhea-galactorrhea due to a pituitary adenoma secreting TSH, TSH-alpha, and PRL. Her serum T4 concentration was 14 micrograms/dl; T3, 5.7 ng/ml, and TSH, 19-33 microU/ml. Serum TSH was not altered by TRH stimulation or T3 suppression. Basal plasma PRL levels were 19-27 ng/ml and plasma PRL doubled after TRH stimulation. A 900-mg pituitary tumor, removed by transphenoidal surgery, was studied in cell culture. After dispersion, tumor cells were maintained on an extracellular matrix produced by bovine corneal endothelial cells in a defined serum-free medium. The hormones released in the culture medium were analyzed by high pressure gel chromatography. Three fractions of tumor TSH were found, with respective apparent mol wts of 45,000 (11%), 28,000 (70%), and 20,000 (19%). Tumoral PRL eluted as a single peak of apparent mol wt of 24,000. Pharmacological studies of TSH, TSH-alpha, and PRL release using thyroid hormones (T3), dopamine agonist (bromocriptine), TRH, and cholera toxin yielded the following results: 1) T3 after 3 days of incubation produced a dose-dependent inhibition of TSH, TSH-alpha, and PRL release. Maximal inhibition (81%) was obtained at 10(-9) M and half-maximal inhibition at 4-6 X 10(-11) M. 2) Bromocriptine produced rapid and partial inhibition of hormone release. Maximal inhibition (51%) was obtained at 10(-8) M and half-maximal inhibition at 5 X 10(-10) M. 3) TRH at 10(-8) M concentration significantly stimulated PRL release but it had no effect on TSH release. 4) Adenylate cyclase activation by 10(-11) M cholera toxin increased TSH (152%), TSH-alpha (150%), and PRL (220%). Immunohistochemical analysis of serial 2 micron sections of the tumor showed that: 1) TSH-alpha immunoreactive cells were the most numerous, 2) TSH-beta positive cells were always positive for TSH-alpha, 3) PRL immunoreactivity was found either uniquely in some cells and colocalized with TSH-alpha immunoreactivity in other cells. However, by electron microscopy, the tumor cells were thyrotrophs. These data indicate that in this patient's tumor: 1) cells secreting TSH were responsive in vitro to near physiological concentrations of thyroid hormones. 2) The colocalization of PRL and TSH-alpha immunoreactivities in some cells raises the possibility either of fusion of differentiated pituitary cells synthesizing distinct hormones or of transformation of less differentiated multipotential pituitary cells.