Highly purified human TSH (hTSH) was dissociated into its alpha and beta subunits with propionic acid; the subunits were separated from undissociated hTSH and;-from each other by gel and ion exchange chromatography. During purification hTSH-a was identified by its cross reaction in a radioimmunoassay for the alpha subunit of human chorionic gonadotropin (hCG-α), and hTSH-β was identified by its cross reaction in an hTSH immunoassay. Purified hTSH-α (> 90% pure) and hTSH-β (>95% pure) were used toinduce antibodies in rabbits by a primary injection of 10 or 15 (µg, respectively, followed by 1 to 3 booster injections of 5 μg. Using hTSH-α tracer and anti-hTSH-α from each of 3 rabbits, radioimmunoassays were developed capable of detecting about 1 ng'ml hTSH-α. In these hTSH-α assays, the cross reactivity of hCG-α and hLH-α was virtually complete, while the cross reactivity of hLH was 15% and the reactivity of hTSH, hTSH-β,and hCG was less than 5%. Using hTSH-β tracer and anti-hTSH-β from each of 3 rabbits, radioimmunoassays were developed capable of detecting 0.025'0.25 ng⁄ml hTSH-β. Two of these immunoassays displayed conventional decreased bindingof tracer with increments of unlabeled hTSH-β, while one showed paradoxical enhanced binding. In these hTSH-β assays, hTSH, hTSH-α, hCG, hCG-α,hCG-β, hLH, and hLH-β displayed cross reactivity of 1% or less. The development of hTSH subunit radioimmunoassays provides additional information about the immunologic relationships of glycoprotein hormones and permits measurement of free subunit concentrations in certain unfractionated human sera. (Endocrinology94: 1411, 1974)