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The Stratus total triiodothyronine (T3) immunoassay is an automated fluorometric enzyme immunoassay that utilizes a mouse monoclonal anti-T3 antibody preimmobilized onto glass fiber paper. The rate of formation of the enzyme product, as measured by front surface fluorometry, is inversely proportional to Total T3 concentration in the sample. The authors evaluated this method with respect to precision, sensitivity, interfering factors, and correlation with a radioimmunoassay. The overall, between-run, and within-run precision of the assay measured at three concentration levels for a total of 60 determinations each using immunoassay control materials, ranged from 2.5% to 14.3%. A total of 200 specimens, including 40 classified as hyperthyroid, and 38 classified as hypothyroid were analyzed in duplicate by the Stratus (STR) system and by a commercially available radioimmunoassay method. The coefficient of correlation obtained was 0.97. Icteric, hemolyzed, azotemic, and lipemic samples were included in the comparison and do not appear to have any interfering effect on the assay. The range of the assay is from 0.8 to 12 nmol/L. In summary, the Stratus Total T3 immunoassay offers the advantages of sensitivity, specificity, and automation with a throughput rate of 45 samples/h.