The beta subunit of TSH (TSH-beta) usually cannot be detected (less than 0.2 ng/ml) in the serum of normal individuals, whereas patients with primary hypothyroidism exhibit elevated TSH-beta levels (0.2-9.3 ng/ml), which increase further after the administration of TRH. Two patients were found to have large TSH-beta as the only form of serum TSH-beta immunoactivity. Patient A was a euthyroid woman with a goiter; TSH and alpha subunit levels were normal (1 microU/ml and 0.6 ng/ml, respectively); TSH-beta was elevated (8-24 ng/ml). Patient B was a woman with borderline hypothyroidism, an elevated serum TSH level (19 microunits/ml), a normal serum alpha level (2.4 ng/ml), and an elevated serum TSH-beta level (1.8-3.6 ng/ml). Dilutions of both patients' sera demonstrated nonparallelism of their serum TSH-beta to standard TSH-beta. The elevated serum TSH-beta levels did not increase after TRH, although TSH and alpha subunit increased appropriately. After the administration of dexamethasone or T4 to patient B, serum TSH-beta did not decrease, although TSH and alpha decreased. Gel chromatography and rechromatography of the patients' sera on a Sephadex G-100 column showed elution of all TSH-beta immunoactivity in or near the void volume (Vo; greater than 150,000 mol wt), whereas sera of hypothyroid patients demonstrated less than 7% of TSH-beta immunoactivity in the Vo. By chromatography on a Sephadex G-200 column, the TSH-beta immunoactivity had a 160,000 mol wt in patient A and 200,000 mol wt in patient B. Incubation of labeled or unlabeled TSH-beta with serum or gamma-globulin fractions from both patients resulted in no significant increase in the binding of TSH-beta to serum components, as determined by both gel chromatography and precipitation with antihuman gamma-globulin. Large TSH-beta was stable after incubation with 6 M guanidine. Ribonuclease failed to affect the large TSH-beta. Inter-chain disulfide bonding was not demonstrated in large TSH-beta after treatment with three different reducing agents (mercaptoethanol, sodium sulfite, and dithioerythritol). Treatment with trypsin did not convert the large TSH-beta immunoactivity to standard TSH-beta. These experiments demonstrated that the large TSH-beta immunoactivity was not caused by binding of TSH-beta to an immunoglobulin or other serum protein or by aggregation of TSH-beta molecules. The significance of these apparently covalently bonded large forms of TSH-beta immunoactivity is not yet known; the presence of small amounts of a large molecular weight form in the serum of hypothyroid patients and normal pituitary extracts raises the possibility that they may be components of normal TSH biosynthesis or represent posttranslational modifications.