Knowledge of the specific activity of the125I-tracer is essential for optimization and for calculation of RIA parameters. The specific activity of the125I-thyroxin used in thyroxin radioimmunoassay /RIA/ has been determined by a simple method involving combination of RIA and displacement analysis. It has been compared with the value obtained by the conventional method based on radioiodination data. Our studies indicate that even for a non-protein hormone like thyroxin the specific activity of125I-thyroxin derived from iodination data is not reliable. The specific activities obtained by displacement analysis were consistent with the experimental findings.