A radioimmunoassay for the measurement of 3,5‐diiodothyronine (3,5‐T2) using a 3H‐labelled derivative of 3,5‐T2 as tracer has been developed. The cross‐reactivity of the antiserum was relatively high for T3 (3.1%) and low for T4 (< 0.0015%). 3,5‐T2 serum concentrations measured after fractional enrichment of 3,5‐T2 were near or below the detection limit of 0.1 nmol/l in ten normal subjects and 0.38 nmol/l in five hyperthyroid patients. 3,5‐T2 was also detected in thyroid tissue. The serum half‐time of 3,5‐T2 estimated after injection of 2 mg 3,5‐T2 in three normal volunteers was 4.5 h. Copyright © 1978, Wiley Blackwell. All rights reserved