Interferon-alpha inducers were previously shown to cause human lymphocyte production of a corticotropin (ACTH)-like peptide. Thyrotropin (TSH) was not produced under these conditions. In contrast, this report shows that a T-cell mitogen (staphylococcal enterotoxin A), which does not induce the ACTH-like peptide, caused human lymphocyte production of an immunoreactive (ir) TSH. Lymphocyte synthesis of the ir TSH was first detectable at 24 hr, peaked at 48 hr, and thereafter declined. NaDodSO4/polyacrylamide gel electrophoresis of intrinsically radiolabeled lymphocyte-derived ir TSH showed radiolabeled peaks at 80, 50, and 26 kilodaltons. These peaks presumably correspond to trimeric, dimeric, and monomeric TSH-like proteins, respectively. Acid treatment and reduction caused the ir TSH to migrate as a 14-kilodalton peak with a 12-kilodalton shoulder in a gel filtration column run in 6 M guanidine . HCl. Thus, the ir TSH seemed to be composed of subunits with molecular masses corresponding to those of the beta and alpha chains of human TSH, respectively. The ir TSH appeared to be glycoprotein because it bound to a concanavalin A affinity column. Taken together these data suggest that in addition to ACTH, human lymphocytes can also produce a TSH-like substance.