A radioiodination technique is described which consists of heating Testosterone (T) with Na125I and hydrogen peroxide in acetic acid in sealed vial. The binding capacity of labelled substances was measured after thin layer chromatography of the reaction mixture. One compound was selected for radioimmunoassay on the basis of its binding capacity for anti-T serum. Standard curves utilizing tritiated and radioiodinated T, under identical conditions, were superimposible. Serum hormone concentrations determined by the two tracers were comparable. Accuracy, precision and sensitivity were satisfactory. Mass spectroscopy and nuclear magnetic resonance of the unlabelled iodinated T indicated that iodine is in C-2.