Primary cultures from three serially transplanted mouse pituitary thyrotropic tumors, Furth 97A, 97B, and 97C, and one newly generated tumor, NIH 101A, were shown to secrete free alpha subunits and a beta subunit (TSH-beta) in addition to intact TSH. The three glycopeptides were measured in specific, heterologous radioimmunoassays. Basal secretion of alpha subunit was found to be in excess in all four thyrotropic tumor cultures, ranging from 5.7- to 8.9-fold higher than that of intact TSH and 30- to 35-fold higher than that of free TSH-beta. In cultures from 97A, TRH (10(-7) M) stimulated the secretion of TSH, alpha subunit, and TSH-beta by 175-185% and (T4, 10(-5) M) inhibited TSH to 19%, alpha subunit to 68%, and TSH-beta to 58% of control. Secretion of TSH and its subunits in cultures from 97B was not consistently affected by TRH or T4. In cultures from 97C, TSH and alpha secretion was inhibited in a dose-dependent fashion by T3 from 2.5 x10(-9) to 1.6x10(-7) M. Gel chromatography of incubation media from tumor 97A revealed elution volumes of TSH and alpha similar to those of purified rat glycopeptides and confirmed the excess secretion of free alpha subunit. In contrast, the alpha to TSH ratios in media from cultures of pituitary cells from normal and thyroidectomized rats were 1.6 and 0.8, respectively. The total production of TSH and its subunits was measured in cultures from tumor 97A and confirmed that alpha subunit was synthesized excessively. The ratio of the plasma concentrations of alpha to TSH in tumor-bearing mice was 1.6 compared to 1.3 in thyroidectomized mice, and 0.36 in thyroidectomized rats. The alpha to TSH ratios in extracts of the corresponding thyrotropic tissues (tumor or pituitary) were 2.7, 1.7, and 0.40, respectively. These data are analogous to those reported in humans with thyrotropic and certain other pituitary tumors, and support hypothesis that the subunits of glycoprotein hormones are independently synthesized.