Repetitive administration of thyrotropin-releasing hormone (TRH) to human subjects was used to produce small elevations of endogenous serum triiodothyronine (T(3)) and thyroxine (T(4)) levels and thereby to determine the effect of these small elevations on the serum thyrotropin (TSH) response to subsequent doses of TRH. Each subject received 13 consecutive doses of 25 mug TRH at 4-h intervals. Serum T(3), T(4), and TSH levels were measured before the 1st, 7th, and 13th doses ("basal levels") and for the 4 h after each of these doses. In 10 normal subjects, the mean TSH response fell from 14.6 muU/ml after the 1st TRH dose to 6.9 and 3.0 muU/ml after the 7th, and 13th doses. These falls in TSH response were accompanied by rises in the mean basal serum T(3) levels from 81 to 115 to 114 ng/100 ml (normal range, 70-150 ng/100 ml) and rises in the mean basal serum T(4) from 6.7 to 8.6 to 9.5 mug/100 ml (normal range, 5-11 mug/100 ml). These data suggest that TRH-induced TSH release is extremely sensitive to inhibition by small elevations, not above the normal ranges, of serum T(3) and T(4) of endogenous origin. In four patients with primary hypothyroidism, the mean TSH responses were 92, 137, and 92 muU/ml after the 1st, 7th, and 13th TRH doses. The corresponding mean basal serum T(3) and T(4) levels at the times of these doses were 34, 30, and 32 ng/100 ml and 1.9, 1.9, and 1.7 mug/100 ml. These data show that repetitive administration of TRH does not result in progressively lower TSH responses in the absence of corresponding increases in serum T(3) and T(4) level. The progressive fall in TSH response observed in the normal subjects, therefore, was apparently due to the corresponding small increases in serum T(3) and T(4) levels and not to progressive depletion of pituitary TSH. In two patients with presumed TRH deficiency, the TSH responses were blunted by repetitive TRH doses but only when the serum T(3) and T(4) levels increased to within the normal ranges. TRH deficiency was thus confirmed for the first time by producing euthyroidism by replacement of TRH.