Circadian rhythm of TSH secretion is characterized by a pronounced nocturnal increment that is not followed by the expected rise of circulating thyroid hormone levels. These findings suggest that the nocturnal TSH surge may be constituted by molecules with reduced bioactivity. We, therefore, investigated TSH bioactivity (measured as cAMP accumulation in FRTL-5 cells) and its carbohydrate structure (by Concanavalin A affinity chromatography) in different blood pools taken during the day and night from seven normal subjects and from one patient with mild (mPH) and five with severe primary hypothyroidism (sPH). Patients with sPH were also studied during low dose L-T4 treatment. Cosinor analysis showed a significant TSH circadian rhythm in the control group and in L-T4-treated sPH patients. The nocturnal TSH surge was not followed by any increase in free thyroid hormone levels. In normal subjects, the daytime ratio of TSH bioactivity to immunoreactivity (TSH B/I) was higher than the nocturnal one [1.4 +/- 0.6 (+/- SD) vs. 1.1 +/- 0.6; P