Iodination of tyrosine residue in subtilisin Carlsberg was studied. Iodination was carried out by lactoperoxidase with various concentrations of K^(125)I to examine the degree of iodine incorporation. Incorporation of iodine was linear up to 0.3 mM KI and saturation occurred above 0.7 mM KI. 7-8 out of 13 tyrosine residues were iodinated. The concentration of KI at which one iodine atom was incorporated was 0.05 mM. At this condition, spectroscopic studies and peptide mapping of iodinated subtilisin were performed. As the degree of iodine incorporation increased, iodinated subtilisin migrated more rapidly in isoelectricfocusing and non-dissociating PAGE. The pH differential spectra of iodinated subtilisin was red-shifted up to 4 nm compared with native one. The pKa of phenolic hydroxyl group of iodinated protein altered from 10.13 to 9.90. Fluorescence of subtilisin at 300 nm decreased when the protein was iodinated. To identify the most reactive tyrosine, iodinated subtilisin was digested with trypsin and resulting peptide of the highest ^(125)I radioactivity was separated by HPLC. From the analysis of amino acid composition, it was suggested that Tyr104 was the likely residue that can be iodinated most easily.