Serum triiodothyronine (T(3)) has been measured by radioimmunoassay and corroborated by analysis of the identical samples with a previously described gas-liquid chromatographic technique. Special features of the radioimmunoassay procedure which permit determinations in unextracted serum include the use of a T(3)-free serum preparation for the construction of the standard curve and of tetrachlorothyronine to inhibit binding of T(3) to thyroxine-binding globulin.T(3) values by radioimmunoassay were 138 +/-23 ng/100 ml (mean +/-SD) in 82 normal subjects, 62 +/-9 ng/100 ml in 45 hypothyroid patients, and 494 +/-265 ng/100 ml in 60 patients with toxic diffuse goiter. In the hypothyroid group, the range was similar in patients with both primary and secondary hypothyroidism. There was no overlap between the three thyroidal states. Elevated T(3) levels were seen in 40 cases that appeared clinically hyperthyroid but had normal serum thyroxine (T(3)) determinations, a syndrome we have called T(3) toxicosis. Values obtained with radioimmunoassay agreed closely with those we had previously found by gas-liquid chromatography which were 68 +/-2 ng/100 ml in hypothyroidism, 137 +/-23 ng/100 ml in normal subjects, and 510 +/-131 ng/100 ml in untreated toxic diffuse goiter. Since T(3) is very potent and its level varies in different clinical states, accurate T(3) measurements are required to assess a patient's thyroid status properly. The radioimmunoassay for T(3) appears to be sufficiently sensitive, precise, and simple to permit its routine clinical application for this purpose.