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 (less than 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.